Secreted and transmembrane polypeptides and nucleic acids encoding the same

ABSTRACT

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides.

BACKGROUND OF THE INVENTION

[0002] Extracellular proteins play important roles in, among otherthings, the formation, differentiation and maintenance of multicellularorganisms. The fate of many individual cells, e.g., proliferation,migration, differentiation, or interaction with other cells, istypically governed by information received from other cells and/or theimmediate environment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiationfactors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

[0003] Secreted proteins have various industrial applications, includingas pharmaceuticals, diagnostics, biosensors and bioreactors. Mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane proteins, also have potential as therapeutic ordiagnostic agents. Efforts are being undertaken by both industry andacademia to identify new, native secreted proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted proteins. Examples ofscreening methods and techniques are described in the literature [see,for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996);U.S. Pat. No. 5,536,637)].

[0004] Membrane-bound proteins and receptors can play important rolesin, among other things, the formation, differentiation and maintenanceof multicellular organisms. The fate of many individual cells, e. g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

[0005] Membrane-bound proteins and receptor molecules have variousindustrial applications, including as pharmaceutical and diagnosticagents. Receptor immunoadhesins, for instance, can be employed astherapeutic agents to block receptor-ligand interactions. Themembrane-bound proteins can also be employed for screening of potentialpeptide or small molecule inhibitors of the relevant receptor/ligandinteraction.

[0006] Efforts are being undertaken by both industry and academia toidentify new, native receptor or membrane-bound proteins. Many effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

[0007] 1. PRO1800

[0008] Hep27 protein is synthesized and accumulated in the nucleus ofhuman hepatoblastoma cells (HepG2 cells) following growth arrest inducedby butyrate treatment (Gabrielli et al., Eur. J. Biochem. 232:473-477(1995)). The synthesis of Hep27 is inhibited in cells that, releasedfrom the butyrate block, have resumed DNA synthesis. The Hep27 proteinsequence shows significant homology to the known short-chain alcoholdehydrogenase (SCAD) family of proteins and it has been suggested thatHep27 is a new member of the SCAD family of proteins. In agreement withits nuclear localization, Hep27 has a region similar to the bipartitenuclear-targeting sequence and Hep27 mRNA expression and proteinsynthesis suggests the existence of a regulation at thepost-transcriptional level.

[0009] We herein describe the identification and characterization ofnovel polypeptides having homology to Hep27 protein, designated hereinas PRO1800 polypeptides.

[0010] 2. PRO539

[0011] Development of multicellular organisms depends, at least in part,on mechanisms which specify, direct or maintain positional informationto pattern cells, tissues, or organs. Various secreted signalingmolecules, such as members of the transforming growth factor-beta(TGF-β), Wnt, fibroblast growth factors and hedgehog families have beenassociated with patterning activity of different cells and structures inDrosophila as well as in vertebrates. Perrimon, Cell 80:517-520 (1995).

[0012] Costal-2 is a novel kinesin-related protein in the Hedgehogsignaling pathway. Hedgehog (Hh) was first identified as asegment-polarity gene by a genetic screen in Drosophila melanogaster,Nusslein-Volhard et al., Roux. Arch. Dev. Biol. 193: 267-282 (1984),that plays a wide variety of developmental functions. Perrimon, supra.Although only one Drosophila Hh gene has been identified, threemammalian Hh homologues have been isolated: Sonic Hh (SHh), Desert Hh(DHh) and Indian Hh (IHh), Echelard et al., Cell 75: 1417-30 (1993);Riddle et al., Cell 75: 1401-16 (1993). SHh is expressed at high levelin the notochord and floor plate of developing vertebrate embryos. Invitro explant assays as well as ectopic expression of SHh in transgenicanimals show that SHh plays a key role in neuronal tube patterning,Echelard et al., supra., Krauss et al., Cell 75, 1432-44 (1993), Riddleet al., Cell 75: 1401-16 (1993), Roelink et al, Cell 81: 445-55 (1995).In vitro explant assays as well as ectopic expression of SHh intransgenic animals show that SHh plays a key role in neural tubepatterning, Echelard et al. (1993), supra.; Ericson et al., Cell 81:747-56 (1995); Marti et al., Nature 375: 322-5 (1995); Roelink et al.(1995), supra; Hynes et al., Neuron 19: 15-26 (1997). Hh also plays arole in the development of limbs (Krauss et al., Cell 75: 1431-44(1993); Laufer et al., Cell 79, 993-1003 (1994)), somites (Fan andTessier-Lavigne, Cell 79, 1175-86 (1994); Johnson et al., Cell 79:1165-73 (1994)), lungs (Bellusci et al., Develop. 124: 53-63 (1997) andskin (Oro et al., Science 276: 817-21 (1997). Likewise, IHh and DHh areinvolved in bone, gut and germinal cell development, Apelqvist et al.,Curr. Biol. 7: 801-4 (1997); Bellusci et al., Dev. Suppl. 124: 53-63(1997); Bitgood et al., Curr. Biol. 6: 298-304 (1996); Roberts et al.,Development 121: 3163-74 (1995). SHh knockout mice further strengthenedthe notion that SHh is critical to many aspect of vertebratedevelopment, Chiang et al., Nature 383: 407-13 (1996). These mice showdefects in midline structures such as the notochord and the floor plate,absence of ventral cell types in neural tube, absence of distal limbstructures, cyclopia, and absence of the spinal column and most of theribs.

[0013] At the cell surface, the Hh signals is thought to be relayed bythe 12 transmembrane domain protein Patched (Ptch) [Hooper and Scott,Cell 59: 751-65 (1989); Nakano et al., Nature 341: 508-13 (1989)] andthe G-protein coupled like receptor Smoothened (Smo) [Alcedo et al.,Cell 86: 221-232 (1996); van den Heuvel and Ingham, Nature 382: 547-551(1996)]. Both genetic and biochemical evidence support a receptor modelwhere Ptch and Smo are part of a multicomponent receptor complex, Chenand Struhl, Cell 87: 553-63 (1996); Marigo et al., Nature 384: 176-9(1996); Stone et al., Nature 384: 129-34 (1996). Upon binding of Hh toPtch, the normal inhibitory effect of Ptch on Smo is relieved, allowingSmo to transduce the Hh signal across the plasma membrane. Loss offunction mutations in the Ptch gene have been identified in patientswith the basal cell nevus syndrome (BCNS), a hereditary diseasecharacterized by multiple basal cell carcinomas (BCCs). DisfunctionalPtch gene mutations have also been associated with a large percentage ofsporadic basal cell carcinoma tumors, Chidambaram et al., CancerResearch 56: 4599-601 (1996); Gailani et al., Nature Genet. 14: 78-81(1996); Hahn et al., Cell 85: 841-51 (1996); Johnson et al., Science272: 1668-71 (1996); Unden et al., Cancer Res. 56: 4562-5; Wicking etal., Am. J. Hum. Genet. 60: 21-6 (1997). Loss of Ptch function isthought to cause an uncontrolled Smo signaling in basal cell carcinoma.Similarly, activating Smo mutations have been identified in sporatic BCCtumors (Xie et al., Nature 391: 90-2 (1998)), emphasizing the role ofSmo as the signaling subunit in the receptor complex for SHh. However,the exact mechanism by which Ptch controls Smo activity still has yet tobe clarified and the signaling mechanisms by which the Hh signal istransmitted from the receptor to downstream targets also remain to beelucidated. Genetic epistatic analysis in Drosophila has identifiedseveral segment-polarity genes which appear to function as components ofthe Hh signal transduction pathway, Ingham, Curr. Opin. Genet. Dev. 5:492-8 (1995); Perrimon, supra. These include a kinesin-like molecule,Costal-2 (Cos-2) [Robbins et al., Cell 90: 225-34 (1997); Sisson et al.,Cell 90: 235-45 (1997)], a protein designated fused [Preat et al.,Genetics 135: 1047-62 (1990); Therond et al., Proc. Natl Acad Sci. USA93: 4224-8 (1996)], a novel molecule with unknown function designatedSuppressor of fused [Pham et al., Genetics 140: 587-98 (1995); Preat,Genetics 132: 725-36 (1992)] and a zinc finger protein Ci. [Alexandre etal., Genes Dev. 10: 2003-13 (1996); Dominguez et al., Science 272:1621-5 (1996); Orenic et al, Genes Dev. 4: 1053-67 (1990)]. Additionalelements implicated in Hh signaling include the transcription factor CBP[Akimaru et al., Nature 386: 735-738 (1997)], the negative regulatorslimb [Jiang and Struhl, Nature 391: 493-496 (1998)] and the SHhresponse element COUP-TFII [Krishnan et al., Science 278: 1947-1950(1997)].

[0014] Mutants in Cos-2 are embryonicly lethal and display a phenotypesimilar to Hh over expression, including duplications of the centralcomponent of each segment and expansion domain of Hh responsive genes.In contrast, mutant embryos for fused and Ci show a phenotype similar toHh loss of function including deletion of the posterior part of eachsegment and replacement of a mirror-like image duplication of theanterior part or each segment and replacement of a mirror-likeduplication of the anterior part, Busson et al., Roux. Arch. Dev. Biol.197: 221-230 (1988). Molecular characterizations of Ci suggested that itis a transcription factor which directly activates Hh responsive genessuch as Wingless and Dpp, Alexandre et al., (1996) supra, Dominguez etal., (1996) supra. Likewise, molecular analysis of fused reveals that itis structurally related to serine threonine kinases and that both intactN-terminal kinase domain and a C-terminal regulatory region are requiredfor its proper function, Preat et al., Nature 347: 87-9 (1990); Robbinset al., (1997), supra; Therond et al., Proc. Natl. Acad. Sci. USA 93:4224-8 (1996). Consistent with the putative opposing functions of Cos-2and fused, fused mutations are suppressed by Cos-2 mutants and also bySuppressor of fused mutants, Preat et al., Genetics 135: 1047-62 (1993).However, whereas fused null mutations and N-terminal kinase domainmutations can be fully suppressed by Suppressor of fused mutations,C-terminus mutations of fused display a strong Cos-2 phenotype in aSuppressor of fused background. This suggests that the fused kinasedomain can act as a constitutive activator of SHh signaling whenSuppressor of Fused is not present. Recent studies have shown that the92 kDa Drosophila used, Cos-2 and Ci are present in a microtubuleassociated multiprotein complex and that Hh signaling leads todissociation of this complex from microtubules, Robbins et al, Cell 90:225-34 (1997); Sisson et al., Cell 90: 235-45 (1997). Both fused andCos-2 become phosphorylated in response to Hh treatment, Robbins et al.,supra; Therond et al., Genetics 142: 1181-98 (1996), but the kinase(s)responsible for this activity(ies) remain to be characterized. To date,the only known vertebrate homologues for these components are members ofthe Gli protein family (e.g., Gli-1, Gli-2 and Gli-3). These are zincfinger putative transcription factors that are structurally related toCi. Among these, Gli-1 was shown to be a candidate mediator of the SHhsignal [Hynes et al., Neuron 15: 35-44 (1995), Lee et al., Development124: 2537-52 (1997); Alexandre et al., Genes Dev. 10: 2003-13 (1996)]suggesting that the mechanism of gene activation in response to Hh maybe conserved between fly and vertebrates. To determine whether othersignaling components in the Hh cascade are evolutionarily conserved andto examine the function of fused in the Hh signaling cascade on thebiochemical level, Applicants have isolated and characterized the humanfused cDNA. Tissue distribution on the mouse indicates that fused isexpressed in SHh responsive tissues. Biochemical studies demonstratethat fused is a functional kinase. Functional studies provide evidencethat fused is an activator of Gli and that a dominant negative form offused is capable of blocking SHh signaling in Xenopus embryos. Togetherthis data demonstrated that both Cos-2 and fused are directly involvedin Hh signaling.

[0015] For additional references related to the Costal-2 protein, seeSimpson et al., Dev. Biol. 122:201-209 (1987), Grau et al., Dev. Biol.122:186-200 (1987), Preat et al., Genetics 135:1047-1062 (1993), Sissonet al., Cell 90:235-245 (1997) and Robbins et al., Cell 90:225-234(1997).

[0016] Applicants have herein identified and describe a cDNA encoding ahuman Costal-2 homolog polypeptide, designated herein as PRO539.

[0017] 3. PRO982

[0018] Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. We herein describe theidentification and characterization of novel secreted polypeptides,designated herein as PRO982 polypeptides.

[0019] 4. PRO1434

[0020] The nel gene has been described to encode a protein that isexpressed in the neural tissues of chicken (Watanabe et al., Genomics38(3):273-276 (1996)). Recently, two novel human cDNAs (designated NELL1and NELL2) have been isolated and characterized which encodepolypeptides having homology to that encoded by the chicken nel gene,wherein those human polypeptides contain six EGF-like repeats (Watanabeet al., supra). Given the neural-specific expression of these genes, itis suggested that they may play a role in neural development. There is,therefore, significant interest in identifying and characterizing novelpolypeptides having homology to nel, NELL1 and NELL2.

[0021] We herein describe the identification and characterization ofnovel polypeptides having homology to the nel protein, designated hereinas PRO1434 polypeptides.

[0022] 5. PRO1863

[0023] Efforts are being undertaken by both industry and academia toidentify new, native transmembrane proteins. Many efforts are focused onthe screening of mammalian recombinant DNA libraries to identify thecoding sequences for novel transmembrane proteins. We herein describethe identification and characterization of novel transmembranepolypeptides, designated herein as PRO1863 polypeptides.

[0024] 6. PRO1917

[0025] The characterization of inositol phosphatases is of interestbecause it is fundamental to the understanding of signaling activitiesthat stimulate the release of Ca²⁺ from the endoplasmic reticulum.Molecular cloning allowed the identification of a multiple inositolpolyphosphate phosphatase which is highly expressed in kidney and liver(Craxton et al. (1997) Biochem J. 328:75-81).

[0026] 7. PRO1868

[0027] The inflammatory response is complex and is mediated by a varietyof signaling molecules produced locally by mast cells, nerve endings,platelets, leucocytes and complement activation. Certain of thesesignaling molecules cause the endothelial cell lining to become moreporous and/or even to express selectins which act as cell surfacemolecules which recognize and attract leucocytes through specificcarbohydrate recognition. Stronger leucocyte binding is mediated byintegrins, which mediate leukocyte movement through the endothelium.Additional signaling molecules act as chemoattractants, causing thebound leucocytes to crawl towards the source of the attractant. Othersignaling molecules produced in the course of an inflammatory responseescape into the blood and stimulate the bone marrow to produce moreleucocytes and release them into the blood stream.

[0028] Inflammation is typically initiated by an antigen, which can bevirtually any molecule capable of initiating an immune response. Undernormal physiological conditions these are foreign molecules, butmolecules generated by the organism itself can serve as the catalyst asis known to occur in various disease states.

[0029] T-cell proliferation is a mixed lymphocyte culture or mixedlymphocyte reaction (MLR) is an established indication of the ability ofa compound to stimulate the immune system. In an inflammatory response,the responding leucocytes can be neutrophilic, eosinophilic, monocyticor lymphocytic. Histological examination of the affected tissuesprovides evidence of an immune stimulating or inhibiting response. SeeCurrent Protocols in Immunology, ed. John E. Coligan, 1994, John Wileyand Sons, Inc.

[0030] Inflammatory bowel disease (IBD) is a term used to collectivelydescribe gut disorders including both ulcerative colitis (UC) andCrohn's disease, both of which are classified as distinct disorders, butshare common features and likely share pathology. The commonality of thediagnostic criteria can make it difficult to precisely determine whichof the two disorders a patient has; however the type and location of thelesion in each are typically different. UC lesions arecharacteristically a superficial ulcer of the mucosa and appear in thecolon, proximal to the rectum. CD lesions are characteristicallyextensive linear fissures, and can appear anywhere in the bowel,occasionally involving the stomach, esophagus and duodenum.

[0031] Conventional treatments for IBD usually involve theadministration of antiinflammatory or immunosuppressive agents, such assulfasalazine, corticosteriods, 6-mercaptopurine/azathoprine, orcyclospoine all of which only bring partial relief to the afflictedpatient. However, when antiinflammatory/immunosuppressive therapiesfail, colectomies are the last line of defense. Surgery is required forabout 30% of CD patients within the first year after diagnosis, with thelikelihood for operative procedure increasing about 5% annuallythereafter. Unfortunately, CD also has a high rate of reoccurrence asabout 5% of patients require subsequent surgery after the initial year.UC patients further have a substantially increased risk of developingcolorectal cancer. Presumably, this is due to the recurrent cycles ofinjury to the epithelium, followed by regrowth, which continuallyincreases the risk of neoplastic transformation.

[0032] A recently discovered member of the immunoglobulin superfamilyknown as Junctional Adhesion Molecule (JAM) has been identified to beselectively concentrated at intercellular junctions of endothelial andepithelial cells of different origins. Martin-Padura, 1. et al., J. CellBiol. 142(1): 117-27 (1998). JAM is a type I integral membrane proteinwith two extracellular, intrachain disulfide loops of the V-type. JAMbears substantial homology to A33 antigen (FIG. 1 or FIG. 18). Amonoclonal antibody directed to JAM was found to inhibit spontaneous andchemokine-induced monocyte transmigration through an endothelial cellmonolayer in vitro. Martin-Padura, supra.

[0033] It has been recently discovered that JAM expression is increasedin the colon of CRF2-4 −/− mice with colitis. CRF 2-4 −/− (IL-10Rsubunit knockout mice) develop a spontaneous colitis mediated bylymphocytes, monocytes and neutrophils. Several of the animals alsodeveloped colon adenocarcinoma. As a result, it is foreseeable likelythat the compounds of the invention are expressed in elevated levels inor otherwise associated with human diseases such as inflammatory boweldisease, other inflammatory diseases of the gut as well as colorectalcarcinoma.

[0034] The compounds of the invention also bear significant homology toA33 antigen, a known colorectal cancer-associated marker. The A33antigen is expressed in more than 90% of primary or metastatic coloncancers as well as normal colon epithelium. In carcinomas originatingfrom the colonic mucosa, the A33 antigen is expressed homogeneously inmore than 95% of all cases. The A33 antigen, however, has not beendetected in a wide range of other normal issues, i.e., its expressionappears to be organ specific. Therefore, the A33 antigen appears to playan important role in the induction of colorectal cancer.

[0035] Since colon cancer is a widespread disease, early diagnosis andtreatment is an important medical goal. Diagnosis and treatment of coloncancer can be implemented using monoclonal antibodies (mAbs) specifictherefore having fluorescent, nuclear magnetic or radioactive tags.Radioactive gene, toxins and/or drug tagged mAbs can be used fortreatment in situ with minimal patient description. mAbs can also beused to diagnose during the diagnosis and treatment of colon cancers.For example, when the serum levels of the A33 antigen are elevated in apatient, a drop of the levels after surgery would indicate the tumorresection was successful. On the other hand, a subsequent rise in serumA33 antigen levels after surgery would indicate that metastases of theoriginal tumor may have formed or that new primary tumors may haveappeared.

[0036] Such monoclonal antibodies can be used in lieu of, or inconjunction with surgery and/or other chemotherapies. For example,preclinical analysis and localization studies in patients infected withcolorectal carcinoma with a mAb to A33 are described in Welt et al., J.Clin. Oncol. 8: 1894-1906 (1990) and Welt et al., J. Clin. Oncol. 12:1561-1571 (1994), while U.S. Pat. No. 4,579,827 and U.S. Ser. No.424,991 (E.P. 199, 141) are directed to the therapeutic administrationof monoclonal antibodies, the latter of which relates to the applicationof anti-A33 mAb.

[0037] We herein describe the identificationand characterization ofnovel polypeptides having homology to A33 antigen protein, designatedherein as PRO1868 polypeptides.

[0038] 8. PRO3434

[0039] Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. We herein describe theidentification and characterization of novel secreted polypeptides,designated herein as PRO3434 polypeptides.

[0040] 9. PRO1927

[0041] Proteins are glycosylated by a complex set of reactions which aremediated by membrane bound glycosyltransferases. There is a large numberof different glycosyltransferases that account for the array ofcarbohydrate structures synthesized. N-acetylglucosaminyltransferaseproteins comprise a family of glycosyltransferases that provide for avariety of important biological functions in the mammalian organism. Asan example, UDP-N-acetylglucosamine: alpha-3-D-mannosidebeta-1,2-N-acetylglucosaminyltransferase I is an glycosyltransferasethat catalyzes an essential first step in the conversion of high-mannoseN-glycans to hybrid and complex N-glycans (Sarkar et al., Proc. Natl.Acad. Sci. USA. 88:234-238 (1991).UPD-N-acetylglucosamine:alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase is an essential enzyme in theproduction of tri- and tetra-antennary asparagine-linked sugar chains,and has been recently been purified from bovine small intestine usingcDNA cloning (Minowa et al., J. Biol. Chem. (1998) 273(19): 11556-62).There is interest in the identification and characterization ofadditional members of the N-acetylglucosaminyltransferase proteinfamily, and more generally, the identification of novelglycosyltransferases.

SUMMARY OF THE INVENTION

[0042] 1. PRO1800

[0043] A cDNA clone (DNA35672-2508) has been identified, having homologyto nucleic acid encoding Hep27 protein, that encodes a novelpolypeptide, designated in the present application as “PRO1800”.

[0044] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1800 polypeptide.

[0045] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO1800 polypeptide having the sequence of aminoacid residues from about 1 or about 16 to about 278, inclusive of FIG. 2(SEQ ID NO:2), or (b) the complement of the DNA molecule of (a).

[0046] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO1800 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about nucleotides 36 orabout 81 and about 869, inclusive, of FIG. 1 (SEQ ID NO:1). Preferably,hybridization occurs under stringent hybridization and wash conditions.

[0047] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203538 (DNA35672-2508) or (b) the complement of the nucleicacid molecule of (a). In a preferred embodiment, the nucleic acidcomprises a DNA encoding the same mature polypeptide encoded by thehuman protein cDNA in ATCC Deposit No. 203538 (DNA35672-2508).

[0048] In still a further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues 1 or about 16 to about 278, inclusive of FIG. 2 (SEQID NO:2), or (b) the complement of the DNA of (a).

[0049] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least 230 nucleotides and produced byhybridizing a test DNA molecule under stringent conditions with (a) aDNA molecule encoding a PRO1800 polypeptide having the sequence of aminoacid residues from 1 or about 16 to about 278, inclusive of FIG. 2 (SEQID NO:2), or (b) the complement of the DNA molecule of (a), and, if theDNA molecule has at least about an 80% sequence identity, prefereably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), isolating the test DNA molecule.

[0050] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1800 polypeptide, with orwithout the N-terminal signal sequence and/or the initiating methionine,or is complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from about aminoacid position 1 to about amino acid position 15 in the sequence of FIG.2 (SEQ ID NO:2).

[0051] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2), or (b) thecomplement of the DNA of (a).

[0052] Another embodiment is directed to fragments of a PRO1800polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments may be from about 20 to about 80 nucleotidesin length, preferably from about 20 to about 60 nucleotides in length,more preferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1).

[0053] In another embodiment, the invention provides isolated PRO1800polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0054] In a specific aspect, the invention provides isolated nativesequence PRO1800 polypeptide, which in certain embodiments, includes anamino acid sequence comprising residues 1 or about 16 to about 278 ofFIG. 2 (SEQ ID NO:2).

[0055] In another aspect, the invention concerns an isolated PRO1800polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2).

[0056] In a further aspect, the invention concerns an isolated PRO1800polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2).

[0057] In yet another aspect, the invention concerns an isolated PRO1800polypeptide, comprising the sequence of amino acid residues 1 or about16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1800antibody. Preferably, the PRO1800 fragment retains a qualitativebiological activity of a native PRO1800 polypeptide.

[0058] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO1800 polypeptide havingthe sequence of amino acid residues from about 1 or about 16 to about278, inclusive of FIG. 2 (SEQ ID NO:3), or (b) the complement of the DNAmolecule of (a), and if the test DNA molecule has at least about an 80%sequence identity, preferably at least about an 85% sequence identity,more preferably at least about a 90% sequence identity, most preferablyat least about a 95% sequence identity to (a) or (b), (ii) culturing ahost cell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

[0059] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1800 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1800 antibody.

[0060] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO1800 polypeptide bycontacting the native PRO1800 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said polypeptide.

[0061] In a still further embodiment, the invention concerns acomposition comprising a PRO1800 polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier.

[0062] 2. PRO539

[0063] A cDNA clone (DNA47465-1561) has been identified, having homologyto nucleic acid encoding Costal-2 protein, that encodes a novelpolypeptide, designated in the present application as “PRO539”.

[0064] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO539 polypeptide.

[0065] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO539 polypeptide having the sequence of amino acidresidues from about 1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7),or (b) the complement of the DNA molecule of (a).

[0066] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO539 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about nucleotides 186 andabout 2675, inclusive, of FIG. 3 (SEQ ID NO:6). Preferably,hybridization occurs under stringent hybridization and wash conditions.

[0067] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203661 (DNA47465-1561) or (b) the complement of the nucleicacid molecule of (a). In a preferred embodiment, the nucleic acidcomprises a DNA encoding the same mature polypeptide encoded by thehuman protein cDNA in ATCC Deposit No. 203661 (DNA47465-1561).

[0068] In still a further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues 1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7),or (b) the complement of the DNA of (a).

[0069] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least 100 nucleotides and produced byhybridizing a test DNA molecule under stringent conditions with (a) aDNA molecule encoding a PRO539 polypeptide having the sequence of aminoacid residues from 1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7), or(b) the complement of the DNA molecule of (a), and, if the DNA moleculehas at least about an 80% sequence identity, prefereably at least aboutan 85% sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), isolating the test DNA molecule.

[0070] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO539 polypeptide, with orwithout the initiating methionine, or is complementary to such encodingnucleic acid molecule.

[0071] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1to about 830, inclusive of FIG. 4 (SEQ ID NO:7), or (b) the complementof the DNA of (a).

[0072] Another embodiment is directed to fragments of a PRO539polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments are from about 20 to about 80 nucleotides inlength, preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 3 (SEQ ID NO:6).

[0073] In another embodiment, the invention provides isolated PRO539polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0074] In a specific aspect, the invention provides isolated nativesequence PRO539 polypeptide, which in certain embodiments, includes anamino acid sequence comprising residues 1 to about 830 of FIG. 4 (SEQ IDNO:7).

[0075] In another aspect, the invention concerns an isolated PRO539polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7).

[0076] In a further aspect, the invention concerns an isolated PRO539polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 toabout 830, inclusive of FIG. 4 (SEQ ID NO:7).

[0077] In yet another aspect, the invention concerns an isolated PRO539polypeptide, comprising the sequence of amino acid residues 1 to about830, inclusive of FIG. 4 (SEQ ID NO:7), or a fragment thereof sufficientto provide a binding site for an anti-PRO539 antibody. Preferably, thePRO539 fragment retains a qualitative biological activity of a nativePRO539 polypeptide.

[0078] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO539 polypeptide havingthe sequence of amino acid residues from about 1 to about 830, inclusiveof FIG. 4 (SEQ ID NO:7), or (b) the complement of the DNA molecule of(a), and if the test DNA molecule has at least about an 80% sequenceidentity, preferably at least about an 85% sequence identity, morepreferably at least about a 90% sequence identity, most preferably atleast about a 95% sequence identity to (a) or (b), (ii) culturing a hostcell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

[0079] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO539 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO539 antibody.

[0080] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO539 polypeptide bycontacting the native PRO539 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said polypeptide. In apreferred embodiment, the biological activity is either binding tomicrotubules or the ability to complex with fused and cubitusinterruptus.

[0081] In a still further embodiment, the invention concerns acomposition comprising a PRO539 polypeptide, or an agonist or antagonistas hereinabove defined, in combination with a pharmaceuticallyacceptable carrier.

[0082] In yet another embodiment, the invention provides for compoundsand methods for developing antagonists against and agonist promotingPRO539 modulation of Hedgehog signaling. In particular, an antagonist ofvertebrate PRO539 which blocks, prevents, inhibits and/or neutralizedthe normal functioning of PRO539 in SH signaling pathway, including bothsmall bioorganic molecules and antisense nucleotides.

[0083] In yet another embodiment, the invention provides foralternatively spliced variants of human PRO539.

[0084] In still yet a further embodiment, the invention provides amethod of screening or assaying for identifying molecules that alter thePRO539 modulation of hedgehog signaling. Preferably, the moleculeseither prevent interaction of PRO539 with its associative complexingproteins (such as fused or cubitus interruptus) or prevent or inhibitdissociation of complexes. The assay comprises the incubation of amixture comprising PRO539 and a substrate with a candidate molecule anddetection of the ability of the candidate molecule to modulate PRO539hedgehog signaling. The screened molecules preferably are small moleculedrug candidates.

[0085] In yet another embodiment, the method relates to a technique ofdiagnosing to determine whether a particular disorder is modulated byhedgehog signaling, comprising:

[0086] (a) culturing test cells or tissues;

[0087] (b) administering a compound which can inhibit PRO539 modulatedhedgehog signaling; and

[0088] (c) determining whether hedgehog signaling is modulated.

[0089] 3. PRO982

[0090] A cDNA clone (DNA57700-1408) has been identified that encodes anovel polypeptide, designated in the present application as “PRO982.”

[0091] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO982 polypeptide.

[0092] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO982 polypeptide having the sequence of amino acidresidues from 1 or about 22 to about 125, inclusive of FIG. 6 (SEQ IDNO:9), or (b) the complement of the DNA molecule of (a).

[0093] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO982 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about residues 89 andabout 400, inclusive, of FIG. 5 (SEQ ID NO:8). Preferably, hybridizationoccurs under stringent hybridization and wash conditions.

[0094] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203583 (DNA57700-1408), or (b) the complement of the DNAmolecule of (a). In a preferred embodiment, the nucleic acid comprises aDNA encoding the same mature polypeptide encoded by the human proteincDNA in ATCC Deposit No. 203583 (DNA57700-1408).

[0095] In a still further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues from 1 or about 22 to about 125, inclusive of FIG. 6(SEQ ID NO:9), or the complement of the DNA of (a).

[0096] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least about 50 nucleotides, and preferably atleast about 100 nucleotides and produced by hybridizing a test DNAmolecule under stringent conditions with (a) a DNA molecule encoding aPRO982 polypeptide having the sequence of amino acid residues from 1 orabout 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or (b) thecomplement of the DNA molecule of (a), and, if the DNA molecule has atleast about an 80% sequence identity, preferably at least about an 85%sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), isolating the test DNA molecule.

[0097] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO982 polypeptide, with orwithout the N-terminal signal sequence and/or the initiating methionine,or is complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from amino acidposition 1 through about amino acid position 21 in the sequence of FIG.6 (SEQ ID NO:9).

[0098] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or (b) thecomplement of the DNA of (a).

[0099] Another embodiment is directed to fragments of a PRO982polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments are from about 20 to about 80 nucleotides inlength, preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length, and mostpreferably from about 20 to about 40 nucleotides in length.

[0100] In another embodiment, the invention provides isolated PRO982polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

[0101] In a specific aspect, the invention provides isolated nativesequence PRO982 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 or about 22 to 125 of FIG. 6 (SEQ IDNO:9).

[0102] In another aspect, the invention concerns an isolated PRO982polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9).

[0103] In a further aspect, the invention concerns an isolated PRO982polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 22 to 125 of FIG. 6 (SEQ ID NO:9).

[0104] In yet another aspect, the invention concerns an isolated PRO982polypeptide, comprising the sequence of amino acid residues 1 or about22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or a fragmentthereof sufficient to provide a binding site for an anti-PRO982antibody. Preferably, the PRO982 fragment retains a qualitativebiological activity of a native PRO982 polypeptide.

[0105] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO982 polypeptide havingthe sequence of amino acid residues from 1 or about 22 to about 125,inclusive of FIG. 6 (SEQ ID NO:9), or (b) the complement of the DNAmolecule of (a), and if the test DNA molecule has at least about an 80%sequence identity, preferably at least about an 85% sequence identity,more preferably at least about a 90% sequence identity, most preferablyat least about a 95% sequence identity to (a) or (b), (ii) culturing ahost cell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

[0106] 4. PRO1434

[0107] A cDNA clone (DNA68818-2536) has been identified, having homologyto nucleic acid encoding nel protein, that encodes a novel polypeptide,designated in the present application as “PRO1434”.

[0108] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1434 polypeptide.

[0109] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO1434 polypeptide having the sequence of aminoacid residues from about 1 or about 28 to about 325, inclusive of FIG. 8(SEQ ID NO:11), or (b) the complement of the DNA molecule of (a).

[0110] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO1434 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about nucleotides 581 orabout 662 and about 1555, inclusive, of FIG. 7 (SEQ ID NO:10).Preferably, hybridization occurs under stringent hybridization and washconditions.

[0111] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203657 (DNA68818-2536) or (b) the complement of the nucleicacid molecule of (a). In a preferred embodiment, the nucleic acidcomprises a DNA encoding the same mature polypeptide encoded by thehuman protein cDNA in ATCC Deposit No. 203657 (DNA68818-2536).

[0112] In still a further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues 1 or about 28 to about 325, inclusive of FIG. 8 (SEQID NO:11), or (b) the complement of the DNA of (a).

[0113] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least 65 nucleotides and produced by hybridizinga test DNA molecule under stringent conditions with (a) a DNA moleculeencoding a PRO1434 polypeptide having the sequence of amino acidresidues from 1 or about 28 to about 325, inclusive of FIG. 8 (SEQ IDNO:11), or (b) the complement of the DNA molecule of (a), and, if theDNA molecule has at least about an 80% sequence identity, prefereably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), isolating the test DNA molecule.

[0114] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1434 polypeptide, with orwithout the N-terminal signal sequence and/or the initiating methionine,and its soluble, i.e., transmembrane domain deleted or inactivatedvariants, or is complementary to such encoding nucleic acid molecule.The signal peptide has been tentatively identified as extending fromabout amino acid position 1 to about amino acid position 27 in thesequence of FIG. 8 (SEQ ID NO:11). The transmembrane domain has beententatively identified as extending from about amino acid position 11 toabout amino acid position 30 in the PRO1434 amino acid sequence (FIG. 8,SEQ ID NO:11).

[0115] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 28 to about 325, inclusive of FIG. 8 (SEQ ID NO:11), or (b) thecomplement of the DNA of (a).

[0116] Another embodiment is directed to fragments of a PRO1434polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments are from about 20 to about 80 nucleotides inlength, preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 7 (SEQ ID NO:10).

[0117] In another embodiment, the invention provides isolated PRO1434polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0118] In a specific aspect, the invention provides isolated nativesequence PRO1434 polypeptide, which in certain embodiments, includes anamino acid sequence comprising residues 1 or about 28 to about 325 ofFIG. 8 (SEQ ID NO:11).

[0119] In another aspect, the invention concerns an isolated PRO1434polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 28 to about 325, inclusive of FIG. 8 (SEQ ID NO:11).

[0120] In a further aspect, the invention concerns an isolated PRO1434polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 28 to about 325, inclusive of FIG. 8 (SEQ ID NO:11).

[0121] In yet another aspect, the invention concerns an isolated PRO1434polypeptide, comprising the sequence of amino acid residues 1 or about28 to about 325, inclusive of FIG. 8 (SEQ ID NO:11), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1434antibody. Preferably, the PRO1434 fragment retains a qualitativebiological activity of a native PRO1434 polypeptide.

[0122] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO1434 polypeptide havingthe sequence of amino acid residues from about 1 or about 28 to about325, inclusive of FIG. 8 (SEQ ID NO:11), or (b) the complement of theDNA molecule of (a), and if the test DNA molecule has at least about an80% sequence identity, preferably at least about an 85% sequenceidentity, more preferably at least about a 90% sequence identity, mostpreferably at least about a 95% sequence identity to (a) or (b), (ii)culturing a host cell comprising the test DNA molecule under conditionssuitable for expression of the polypeptide, and (iii) recovering thepolypeptide from the cell culture.

[0123] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1434 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1434 antibody.

[0124] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO1434 polypeptide bycontacting the native PRO1434 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said polypeptide.

[0125] In a still further embodiment, the invention concerns acomposition comprising a PRO1434 polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier.

[0126] 5. PRO1863

[0127] A cDNA clone (DNA59847-2510) has been identified that encodes anovel transmembrane polypeptide, designated in the present applicationas “PRO1863”.

[0128] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1863 polypeptide.

[0129] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO1863 polypeptide having the sequence of aminoacid residues from about 1 or about 16 to about 437, inclusive of FIG.10 (SEQ ID NO:16), or (b) the complement of the DNA molecule of (a).

[0130] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO1863 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about nucleotides 17 orabout 62 and about 1327, inclusive, of FIG. 9 (SEQ ID NO:15).Preferably, hybridization occurs under stringent hybridization and washconditions.

[0131] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203576 (DNA59847-2510) or (b) the complement of the nucleicacid molecule of (a). In a preferred embodiment, the nucleic acidcomprises a DNA encoding the same mature polypeptide encoded by thehuman protein cDNA in ATCC Deposit No. 203576 (DNA59847-2510).

[0132] In still a further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues 1 or about 16 to about 437, inclusive of FIG. 10(SEQ ID NO:16), or (b) the complement of the DNA of (a).

[0133] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least 345 nucleotides and produced byhybridizing a test DNA molecule under stringent conditions with (a) aDNA molecule encoding a PRO1863 polypeptide having the sequence of aminoacid residues from 1 or about 16 to about 437, inclusive of FIG. 10 (SEQID NO:16), or (b) the complement of the DNA molecule of (a), and, if theDNA molecule has at least about an 80% sequence identity, prefereably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), isolating the test DNA molecule.

[0134] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1863 polypeptide, with orwithout the N-terminal signal sequence and/or the initiating methionine,and its soluble, i.e., transmembrane domain deleted or inactivatedvariants, or is complementary to such encoding nucleic acid molecule.The signal peptide has been tentatively identified as extending fromabout amino acid position 1 to about amino acid position 17 in thesequence of FIG. 10 (SEQ ID NO:16). The transmembrane domain has beententatively identified as extending from about amino acid position 243to about amino acid position 260 in the PRO1863 amino acid sequence(FIG. 10, SEQ ID NO:16).

[0135] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 16 to about 437, inclusive of FIG. 10 (SEQ ID NO:16), or (b)the complement of the DNA of (a).

[0136] Another embodiment is directed to fragments of a PRO1863polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments are from about 20 to about 80 nucleotides inlength, preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 9 (SEQ ID NO:15).

[0137] In another embodiment, the invention provides isolated PRO1863polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0138] In a specific aspect, the invention provides isolated nativesequence PRO1863 polypeptide, which in certain embodiments, includes anamino acid sequence comprising residues 1 or about 16 to about 437 ofFIG. 10 (SEQ ID NO:16).

[0139] In another aspect, the invention concerns an isolated PRO1863polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 16 to about 437, inclusive of FIG. 10 (SEQ ID NO:16).

[0140] In a further aspect, the invention concerns an isolated PRO1863polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 16 to about 437, inclusive of FIG. 10 (SEQ ID NO:16).

[0141] In yet another aspect, the invention concerns an isolated PRO1863polypeptide, comprising the sequence of amino acid residues 1 or about16 to about 437, inclusive of FIG. 10 (SEQ ID NO:16), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1863antibody. Preferably, the PRO1863 fragment retains a qualitativebiological activity of a native PRO1863 polypeptide.

[0142] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO1863 polypeptide havingthe sequence of amino acid residues from about 1 or about 16 to about437, inclusive of FIG. 10 (SEQ ID NO:16), or (b) the complement of theDNA molecule of (a), and if the test DNA molecule has at least about an80% sequence identity, preferably at least about an 85% sequenceidentity, more preferably at least about a 90% sequence identity, mostpreferably at least about a 95% sequence identity to (a) or (b), (ii)culturing a host cell comprising the test DNA molecule under conditionssuitable for expression of the polypeptide, and (iii) recovering thepolypeptide from the cell culture.

[0143] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1863 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1863 antibody.

[0144] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO1863 polypeptide bycontacting the native PRO1863 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said polypeptide.

[0145] In a still further embodiment, the invention concerns acomposition comprising a PRO1863 polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier.

[0146] 6. PRO1917

[0147] A cDNA clone (DNA76400-2528) has been identified that encodes anovel polypeptide having homology to inositol phosphatase and designatedin the present application as “PRO1917”.

[0148] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1917 polypeptide.

[0149] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO1917 polypeptide having the sequence of aminoacid residues from 1 or about 31 to about 487, inclusive of FIG. 12 (SEQID NO:18), or (b) the complement of the DNA molecule of (a).

[0150] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO1917 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about residues 96 andabout 1466, inclusive, of FIG. 11 (SEQ ID NO:17). Preferably,hybridization occurs under stringent hybridization and wash conditions.

[0151] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203573 (DNA76400-2528), or (b) the complement of the DNAmolecule of (a). In a preferred embodiment, the nucleic acid comprises aDNA encoding the same mature polypeptide encoded by the human proteincDNA in ATCC Deposit No. 203573 (DNA76400-2528).

[0152] In a still further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues from 1 or about 31 to about 487, inclusive of FIG.12 (SEQ ID NO:18), or the complement of the DNA of (a).

[0153] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least about 50 nucleotides, and preferably atleast about 100 nucleotides and produced by hybridizing a test DNAmolecule under stringent conditions with (a) a DNA molecule encoding aPRO1917 polypeptide having the sequence of amino acid residues from 1 orabout 31 to about 487, inclusive of FIG. 12 (SEQ ID NO:18), or (b) thecomplement of the DNA molecule of (a), and, if the DNA molecule has atleast about an 80% sequence identity, preferably at least about an 85%sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), isolating the test DNA molecule.

[0154] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1917 polypeptide, with orwithout the N-terminal signal sequence and/or the initiating methionine,or is complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from amino acidposition 1 through about amino acid position 30 in the sequence of FIG.12 (SEQ ID NO:18).

[0155] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 31 to about 487, inclusive of FIG. 12 (SEQ ID NO:18), or (b)the complement of the DNA of (a).

[0156] Another embodiment is directed to fragments of a PRO1917polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments are from about 20 to about 80 nucleotides inlength, preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length, and mostpreferably from about 20 to about 40 nucleotides in length.

[0157] In another embodiment, the invention provides isolated PRO1917polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

[0158] In a specific aspect, the invention provides isolated nativesequence PRO1917 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 or about 31 to 487 of FIG. 12 (SEQID NO:18).

[0159] In another aspect, the invention concerns an isolated PRO1917polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 31 to about 487, inclusive of FIG. 12 (SEQ ID NO:18).

[0160] In a further aspect, the invention concerns an isolated PRO1917polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 31 to 487 of FIG. 12 (SEQ ID NO:18).

[0161] In yet another aspect, the invention concerns an isolated PRO1917polypeptide, comprising the sequence of amino acid residues 1 or about31 to about 487, inclusive of FIG. 12 (SEQ ID NO:18), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1917antibody. Preferably, the PRO1917 fragment retains a qualitativebiological activity of a native PRO1917 polypeptide.

[0162] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO1917 polypeptide havingthe sequence of amino acid residues from 1 or about 31 to about 487,inclusive of FIG. 12 (SEQ ID NO:18), or (b) the complement of the DNAmolecule of (a), and if the test DNA molecule has at least about an 80%sequence identity, preferably at least about an 85% sequence identity,more preferably at least about a 90% sequence identity, most preferablyat least about a 95% sequence identity to (a) or (b), (ii) culturing ahost cell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

[0163] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1917 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1917 antibody.

[0164] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO1917 polypeptide, bycontacting the native PRO1917 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said polypeptide.

[0165] In a still further embodiment, the invention concerns acomposition comprising a PRO1917 polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier.

[0166] 7. PRO1868

[0167] The present invention concerns compositions and methods for thediagnosis and treatment of inflammatory diseases in mammals, includinghumans. The present invention is based on the identification of proteins(including agonist and antagonist antibodies) which either stimulate orinhibit the immune response in mammals. Inflammatory diseases can betreated by suppressing the inflammatory response. Molecules that enhancean inflammatory response stimulate or potentiate the immune response toan antigen. Molecules which stimulate an inflammatory response can beinhibited where suppression of the inflammatory response would bebeneficial. Molecules which stimulate the inflammatory response can beused therapeutically where enhancement of the inflammatory responsewould be beneficial. Such stimulatory molecules can also be inhibitedwhere suppression of the inflammatory response would be of value.Neutralizing antibodies are examples of molecules that inhibit moleculeshaving immune stimulatory activity and which would be beneficial in thetreatment of inflammatory diseases. Molecules which inhibit theinflammatory response can also be utilized (proteins directly or via theuse of antibody agonists) to inhibit the inflammatory response and thusameliorate inflammatory diseases.

[0168] Accordingly, the proteins of the invention are useful for thediagnosis and/or treatment (including prevention) of immune relateddiseases. Antibodies which bind to stimulatory proteins are useful tosuppress the inflammatory response. Antibodies which bind to inhibitoryproteins are useful to stimulate inflammatory response and the immunesystem. The proteins and antibodies of the invention are also useful toprepare medicines and medicaments for the treatment of inflammatory andimmune related diseases.

[0169] In one embodiment, the invention concerns antagonists andagonists of a PRO1868 polypeptide that inhibits one or more of thefunctions or activities of a PRO1868 polypeptide.

[0170] In another embodiment, the invention concerns a method fordetermining the presence of a PRO1868 polypeptide comprising exposing acell suspected of containing the polypeptide to an anti-PRO1868 antibodyand determining binding of the antibody to the cell.

[0171] In yet another embodiment, the present invention relates to amethod of diagnosing an inflammatory related disease in a mammal,comprising detecting the level of expression of a gene encoding aPRO1868 polypeptide (a) in a test sample of tissue cells obtained fromthe mammal, and (b) in a control sample of known normal tissue cells ofthe same cell type, wherein a higher expression level in the test sampleindicates the presence of an inflammatory disease in the mammal.

[0172] In another embodiment, the present invention relates to method ofdiagnosing an inflammatory disease in a mammal, comprising (a)contacting an anti-PRO1868 antibody with a test sample of tissue culturecells obtained from the mammal, and (b) detecting the formation of acomplex between the antibody and the PRO1868 polypeptide. The detectionmay be qualitative or quantitative, and may be performed in comparisonwith monitoring the complex formation in a control sample of knownnormal tissue cells of the same cell type. A larger quantity ofcomplexes formed in the test sample indicates the presence of tumor inthe mammal from which the test tissue cells were obtained. The antibodypreferably carries a detectable label. Complex formation can bemonitored, for example, by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. The test sample isusually obtained from an individual suspected of having a deficiency orabnormality relating to the inflammatory response.

[0173] In another embodiment, the present invention relates to adiagnostic kit, containing an anti-PRO1868 antibody and a carrier (e.g.,a buffer) in suitable packaging. The kit preferably containsinstructions for using the antibody to detect the PRO1868 polypeptide.

[0174] In a further embodiment, the invention concerns an article ofmanufacture, comprising:

[0175] a container;

[0176] a label on the container; and

[0177] a composition comprising an active agent contained within thecontainer; wherein the composition is effective for stimulating orinhibiting an inflammatory response in a mammal, the label on thecontainer indicates that the composition can be used to treat aninflammatory disease, and the active agent in the composition is anagent stimulating or inhibiting the expression and/or activity of thePRO1868 polypeptide. In a preferred aspect, the active agent is aPRO1868 polypeptide or an anti-PRO1868 antibody.

[0178] A further embodiment is a method for identifying a compoundcapable of inhibiting the expression and/or activity of a PRO1868polypeptide by contacting a candidate compound with a PRO1868polypeptide under conditions and for time sufficient to allow these twocompounds to interact. In a specific aspect, either the candidatecompound or the PRO1868 polypeptide is immobilized on a solid support.In another aspect, the non-immobilized component carries a detectablelabel.

[0179] In yet a further aspect, the invention relates to a method oftreating an inflammatory disease, by administration of an effectivetherapeutic amount of a PRO1868 antagonist to a patient in need thereoffor the treatment of a disease selected from: inflammatory boweldisease, systemic lupus erythematosis, rheumatoid arthritis, juvenilechronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjogren's syndrome, systemic vaculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis ,tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic polyneuropathy, hepatobiliarydiseases such as infectious hepatitis (hepatitis A, B, C, D, E and othernonhepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases (e.g., cystic fibrosis,eosinophilic pneumonias, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis), gluten-sensitive enteropathy, Whipple'sdisease, autoimmune or immune-mediated skin diseases including bullousskin diseases, erythema multiforme and contact dermatitis, psoriasis,allergic diseases of the lung such as eosinophilic pneumonias,idiopathic pulmonary fibrosis and hypersensitivity pneumonitis,transplantation associated diseases including graft rejection andgraft-verus host disease.

[0180] In a further embodiment, the present invention provides a methodof diagnosing tumor in a mammal, comprising detecting the level ofexpression of a gene encoding a PRO1868 polypeptide (a) in a test sampleof tissue cells obtained from the mammal, and (b) in a control sample ofknown normal tissue cells of the same cell type, wherein a higherexpression level in the test sample indicates the presence of tumor inthe mammal from which the test tissue cells were obtained.

[0181] In another embodiment, the present invention provides a method ofdiagnosing tumor in a mammal, comprising (a) contacting an anti-PRO1868antibody with a test sample of the tissue cells obtained from themammal, and (b) detecting the formation of a complex between theanti-PRO1868 and the PRO1868 polypeptide in the test sample. Thedetection may be qualitative or quantitative, and may be performed incomparison with monitoring the complex formation in a control sample ofknown normal tissue cells of the same cell type. A larger quantity ofcomplexes formed in the test sample indicates the presence of tumor inthe mammal from which the test tissue cells were obtained. The antibodypreferably carries a detectable label. Complex formation can bemonitored, for example, by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. Preferably, the testsample is obtained from an individual mammal suspected to haveneoplastic cell growth or proliferation (e.g., cancerous cells).

[0182] In another embodiment, the present invention provides a cancerdiagnostic kit, comprising an anti-PRO1868 antibody and a carrier (e.g.a buffer) in suitable packaging. The kit preferably containsinstructions for using the antibody to detect the PRO1868 polypeptide.

[0183] In yet another embodiment, the invention provides a method forinhibiting the growth of tumor cells comprising exposing a cell whichoverexpresses a PRO1868 polypeptide to an effective amount of an agentinhibiting the expression and/or activity of the PRO1868 polypeptide.The agent preferably is an anti-PRO1868 polypeptide, a small organic andinorganic peptide, phosphopeptide, antisense or ribozyme molecule, or atriple helix molecule. In a specific aspect, the agent, e.g.,anti-PRO1868 antibody induces cell death. In a further aspect, the tumorcells are further exposed to radiation treatment and/or a cytotoxic orchemotherapeutic agent.

[0184] In a further embodiment, the invention concerns an article ofmanufacture, comprising:

[0185] a container;

[0186] a label on the container, and

[0187] a composition comprising an active agent contained within thecontainer; wherein the composition is effective for inhibiting thegrowth of tumor cells, the label on the container indicates that thecomposition can be used for treating conditions characterized byoverexpression of a PRO1868 polypeptide, and the active agent in thecomposition is an agent inhibiting the expression and/or activity of thePRO1868 polypeptide. In a preferred aspect, the active agent is ananti-PRO1868 antibody.

[0188] A cDNA clone (DNA77624-2515) has been identified, having homologyto nucleic acid encoding A33 antigen, that encodes a novel polypeptide,designated in the present application as “PRO1868”.

[0189] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1868 polypeptide.

[0190] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO1868 polypeptide having the sequence of aminoacid residues from about 1 or about 31 to about 310, inclusive of FIG.14 (SEQ ID NO:20), or (b) the complement of the DNA molecule of (a).

[0191] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO1868 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about nucleotides 51 orabout 141 and about 980, inclusive, of FIG. 13 (SEQ ID NO:19).Preferably, hybridization occurs under stringent hybridization and washconditions.

[0192] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203553 (DNA77624-2515) or (b) the complement of the nucleicacid molecule of (a). In a preferred embodiment, the nucleic acidcomprises a DNA encoding the same mature polypeptide encoded by thehuman protein cDNA in ATCC Deposit No. 203553 (DNA77624-2515).

[0193] In still a further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues 1 or about 31 to about 310 inclusive of FIG. 14 (SEQID NO:20), or (b) the complement of the DNA of (a).

[0194] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least 390 nucleotides and produced byhybridizing a test DNA molecule under stringent conditions with (a) aDNA molecule encoding a PRO1868 polypeptide having the sequence of aminoacid residues from 1 or about 31 to about 310, inclusive of FIG. 14 (SEQID NO:20), or (b) the complement of the DNA molecule of (a), and, if theDNA molecule has at least about an 80% sequence identity, prefereably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), isolating the test DNA molecule.

[0195] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1868 polypeptide, with orwithout the N-terminal signal sequence and/or the initiating methionine,and its soluble, i.e., transmembrane domain deleted or inactivatedvariants, or is complementary to such encoding nucleic acid molecule.The signal peptide has been tentatively identified as extending fromabout amino acid position 1 to about amino acid position 30 in thesequence of FIG. 14 (SEQ ID NO:20). The transmembrane domain has beententatively identified as extending from about amino acid position 243to about amino acid position 263 in the PRO1868 amino acid sequence(FIG. 14, SEQ ID NO:20).

[0196] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 31 to about 310 inclusive of FIG. 14 (SEQ ID NO:20), or (b) thecomplement of the DNA of (a).

[0197] Another embodiment is directed to fragments of a PRO1868polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments are from about 20 to about 80 nucleotides inlength, preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length and mostpreferably from about 20 to about 40 nucleotides in length and may bederived from the nucleotide sequence shown in FIG. 13 (SEQ ID NO:19).

[0198] In another embodiment, the invention provides isolated PRO1868polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0199] In a specific aspect, the invention provides isolated nativesequence PRO1868 polypeptide, which in certain embodiments, includes anamino acid sequence comprising residues 1 or about 31 to about 310 ofFIG. 14 (SEQ ID NO:20).

[0200] In another aspect, the invention concerns an isolated PRO1868polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 31 to about 310, inclusive of FIG. 14 (SEQ ID NO:20).

[0201] In a further aspect, the invention concerns an isolated PRO1868polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 31 to about 310, inclusive of FIG. 14 (SEQ ID NO:20).

[0202] In yet another aspect, the invention concerns an isolated PRO1868polypeptide, comprising the sequence of amino acid residues 1 or about31 to about 310, inclusive of FIG. 14 (SEQ ID NO:20), or a fragmentthereof sufficient to provide a binding site for an anti-PRO1868antibody. Preferably, the PRO1868 fragment retains a qualitativebiological activity of a native PRO1868 polypeptide.

[0203] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO1868 polypeptide havingthe sequence of amino acid residues from about 1 or about 31 to about310, inclusive of FIG. 14 (SEQ ID NO:20), or (b) the complement of theDNA molecule of (a), and if the test DNA molecule has at least about an80% sequence identity, preferably at least about an 85% sequenceidentity, more preferably at least about a 90% sequence identity, mostpreferably at least about a 95% sequence identity to (a) or (b), (ii)culturing a host cell comprising the test DNA molecule under conditionssuitable for expression of the polypeptide, and (iii) recovering thepolypeptide from the cell culture.

[0204] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1868 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1868 antibody.

[0205] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO1868 polypeptide bycontacting the native PRO1868 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said polypeptide.

[0206] In a still further embodiment, the invention concerns acomposition comprising a PRO1868 polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier.

[0207] In another embodiment, the invention provides a compositioncontaining a PRO1868 polypeptide or an agonist or antagonist antibody inadmixture with a carrier or excipient. In one aspect, the compositioncontains a therapeutically affective amount of the peptide or antibody.In another aspect, when the composition contains an inflammationstimulating molecule, the composition is useful for: (a) increasinginfiltration of inflammatory cells into a tissue of a mammal in needthereof, (b) stimulating or enhancing an immune response in a mammal inneed thereof, or (c) increasing the proliferation of T-lymphocytes in amammal in need thereof in response to an antigen. In a further aspect,when the composition contains an inflammatory inhibiting molecule, thecomposition is useful for: (a) decreasing infiltration of inflammatorycells into a tissue of a mammal in need thereof, (b) inhibiting orreducing an inflammatory response in a mammal in need thereof, or (c)decreasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen. In another aspect, the compositioncontains a further active ingredient, which may, for example, be afurther antibody or a cytotoxic or chemotherapeutic agent. Preferably,the composition is sterile.

[0208] In a further embodiment, the invention concerns nucleic acidencoding an anti-PRO1868 antibody, and vectors and recombinant hostcells comprising such nucleic acid. In a still further embodiment, theinvention concerns a method for producing such an antibody by culturinga host cell transformed with nucleic acid encoding the antibody underconditions such that the antibody is expressed, and recovering theantibody from the cell culture.

[0209] 8. PRO3434

[0210] A cDNA clone (DNA77631-2537) has been identified that encodes anovel polypeptide, designated in the present application as “PRO3434.”

[0211] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO3434 polypeptide.

[0212] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO3434 polypeptide having the sequence of aminoacid residues from 1 or about 17 to about 1029, inclusive of FIG. 16(SEQ ID NO:22), or (b) the complement of the DNA molecule of (a).

[0213] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO3434 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about residues 46 or about94 and about 3132, inclusive, of FIG. 15 (SEQ ID NO:21). Preferably,hybridization occurs under stringent hybridization and wash conditions.

[0214] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203651 (DNA77631-2537), or (b) the complement of the DNAmolecule of (a). In a preferred embodiment, the nucleic acid comprises aDNA encoding the same mature polypeptide encoded by the human proteincDNA in ATCC Deposit No. 203651 (DNA77631-2537).

[0215] In a still further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues from 1 or about 17 to about 1029, inclusive of FIG.16 (SEQ ID NO:22), or the complement of the DNA of (a).

[0216] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least about 460 nucleotides and produced byhybridizing a test DNA molecule under stringent conditions with (a) aDNA molecule encoding a PRO3434 polypeptide having the sequence of aminoacid residues from 1 or about 17 to about 1029, inclusive of FIG. 16(SEQ ID NO:22), or (b) the complement of the DNA molecule of (a), and,if the DNA molecule has at least about an 80% sequence identity,preferably at least about an 85% sequence identity, more preferably atleast about a 90% sequence identity, most preferably at least about a95% sequence identity to (a) or (b), isolating the test DNA molecule.

[0217] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO3434 polypeptide, with orwithout the N-terminal signal sequence and/or the initiating methionine,or is complementary to such encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from amino acidposition 1 through about amino acid position 16 in the sequence of FIG.16 (SEQ ID NO:22).

[0218] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22), or (b)the complement of the DNA of (a).

[0219] Another embodiment is directed to fragments of a PRO3434polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments are from about 20 to about 80 nucleotides inlength, preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length, and mostpreferably from about 20 to about 40 nucleotides in length.

[0220] In another embodiment, the invention provides isolated PRO3434polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

[0221] In a specific aspect, the invention provides isolated nativesequence PRO3434 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 or about 17 to 1029 of FIG. 16 (SEQID NO:22).

[0222] In another aspect, the invention concerns an isolated PRO3434polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22).

[0223] In a further aspect, the invention concerns an isolated PRO3434polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 17 to 1029 of FIG. 16 (SEQ ID NO:22).

[0224] In yet another aspect, the invention concerns an isolated PRO3434polypeptide, comprising the sequence of amino acid residues 1 or about17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22), or a fragmentthereof sufficient to provide a binding site for an anti-PRO3434tibody.Preferably, the PRO982 fragment retains a qualitative biologicalactivity of a native PRO3434 polypeptide.

[0225] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO3434 polypeptide havingthe sequence of amino acid residues from 1 or about 17 to about 1029,inclusive of FIG. 16 (SEQ ID NO:22), or (b) the complement of the DNAmolecule of (a), and if the test DNA molecule has at least about an 80%sequence identity, preferably at least about an 85% sequence identity,more preferably at least about a 90% sequence identity, most preferablyat least about a 95% sequence identity to (a) or (b), (ii) culturing ahost cell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

[0226] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO3434 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO3434 antibody.

[0227] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO3434 polypeptide, bycontacting the native PRO3434 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said polypeptide.

[0228] In a still further embodiment, the invention concerns acomposition comprising a PRO3434 polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier.

[0229] 9. PRO1927

[0230] A cDNA clone (DNA82307-2531)has been identified that encodes anovel polypeptide having homology to glycosyltransferases, and isdesignated in the present application as “PRO1927”.

[0231] In one embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1927 polypeptide.

[0232] In one aspect, the isolated nucleic acid comprises DNA having atleast about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to (a) a DNAmolecule encoding a PRO1927 polypeptide having the sequence of aminoacid residues from 1 or about 24 to about 548, inclusive of FIG. 18 (SEQID NO:24), or (b) the complement of the DNA molecule of (a).

[0233] In another aspect, the invention concerns an isolated nucleicacid molecule encoding a PRO1927 polypeptide comprising DNA hybridizingto the complement of the nucleic acid between about residues 120 andabout 1694, inclusive, of FIG. 17 (SEQ ID NO:23). Preferably,hybridization occurs under stringent hybridization and wash conditions.

[0234] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising DNA having at least about 80% sequenceidentity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding thesame mature polypeptide encoded by the human protein cDNA in ATCCDeposit No. 203537 (DNA82307-2531), or (b) the complement of the DNAmolecule of (a). In a preferred embodiment, the nucleic acid comprises aDNA encoding the same mature polypeptide encoded by the human proteincDNA in ATCC Deposit No. 203537 (DNA82307-2531).

[0235] In a still further aspect, the invention concerns an isolatednucleic acid molecule comprising (a) DNA encoding a polypeptide havingat least about 80% sequence identity, preferably at least about 85%sequence identity, more preferably at least about 90% sequence identity,most preferably at least about 95% sequence identity to the sequence ofamino acid residues from 1 or about 24 to about 548, inclusive of FIG.18 (SEQ ID NO:24), or the complement of the DNA of (a).

[0236] In a further aspect, the invention concerns an isolated nucleicacid molecule having at least about 50 nucleotides, and preferably atleast about 100 nucleotides and produced by hybridizing a test DNAmolecule under stringent conditions with (a) a DNA molecule encoding aPRO1927 polypeptide having the sequence of amino acid residues from 1 orabout 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or (b) thecomplement of the DNA molecule of (a), and, if the DNA molecule has atleast about an 80% sequence identity, preferably at least about an 85%sequence identity, more preferably at least about a 90% sequenceidentity, most preferably at least about a 95% sequence identity to (a)or (b), isolating the test DNA molecule.

[0237] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a PRO1927 polypeptide, with orwithout the N-terminal signal sequence and/or the initiating methionine,and its soluble variants (i.e. transmembrane domain deleted orinactivated), or is complementary to such encoding nucleic acidmolecule. The signal peptide has been tentatively identified asextending from amino acid position 1 through about amino acid position23 in the sequence of FIG. 18 (SEQ ID NO:24). A type II transmembranedomain has been tentatively identified as extending from about aminoacid position 6 to about amino acid position 25 in the PRO1927 aminoacid sequence (FIG. 18, SEQ ID NO:24).

[0238] In another aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1or about 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or (b)the complement of the DNA of (a).

[0239] Another embodiment is directed to fragments of a PRO1927polypeptide coding sequence that may find use as hybridization probes.Such nucleic acid fragments are from about 20 to about 80 nucleotides inlength, preferably from about 20 to about 60 nucleotides in length, morepreferably from about 20 to about 50 nucleotides in length, and mostpreferably from about 20 to about 40 nucleotides in length.

[0240] In another embodiment, the invention provides isolated PRO1927polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

[0241] In a specific aspect, the invention provides isolated nativesequence PRO1927 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 or about 24 to 548 of FIG. 18 (SEQID NO:24).

[0242] In another aspect, the invention concerns an isolated PRO1927polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 or about 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24).

[0243] In a further aspect, the invention concerns an isolated PRO1927polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 orabout 24 to 548 of FIG. 18 (SEQ ID NO:24).

[0244] In yet another aspect, the invention concerns an isolated PRO1927 polypeptide, comprising the sequence of amino acid residues 1 orabout 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or afragment thereof sufficient to provide a binding site for ananti-PRO1927 antibody. Preferably, the PRO1927 fragment retains aqualitative biological activity of a native PRO1927 polypeptide.

[0245] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a PRO1927 polypeptide havingthe sequence of amino acid residues from 1 or about 24 to about 548,inclusive of FIG. 18 (SEQ ID NO:24), or (b) the complement of the DNAmolecule of (a), and if the test DNA molecule has at least about an 80%sequence identity, preferably at least about an 85% sequence identity,more preferably at least about a 90% sequence identity, most preferablyat least about a 95% sequence identity to (a) or (b), (ii) culturing ahost cell comprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

[0246] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO1927 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-PRO1927 antibody.

[0247] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists of a native PRO1927 polypeptide, bycontacting the native PRO1927 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said polypeptide.

[0248] In a still further embodiment, the invention concerns acomposition comprising a PRO1927 polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier

[0249] 10. Additional Embodiments

[0250] In other embodiments of the present invention, the inventionprovides vectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

[0251] In other embodiments, the invention provides chimericmoleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

[0252] In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

[0253] In yet other embodiments, the invention provides oligonucleotideprobes useful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

[0254] In other embodiments, the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes a PROpolypeptide.

[0255] In one aspect, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculeencoding a PRO polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

[0256] In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculecomprising the coding sequence of a full-length PRO polypeptide cDNA asdisclosed herein, the coding sequence of a PRO polypeptide lacking thesignal peptide as disclosed herein, the coding sequence of anextracellular domain of a transmembrane PRO polypeptide, with or withoutthe signal peptide, as disclosed herein or the coding sequence of anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein, or (b) the complement of the DNA moleculeof (a).

[0257] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) a DNAmolecule that encodes the same mature polypeptide encoded by any of thehuman protein cDNAs deposited with the ATCC as disclosed herein, or (b)the complement of the DNA molecule of (a).

[0258] Another aspect the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated, or is complementary to such encoding nucleotidesequence, wherein the transmembrane domain(s) of such polypeptide aredisclosed herein. Therefore, soluble extracellular domains of the hereindescribed PRO polypeptides are contemplated.

[0259] Another embodiment is directed to fragments of a PRO polypeptidecoding sequence, or the complement thereof, that may find use as, forexample, hybridization probes, for encoding fragments of a PROpolypeptide that may optionally encode a polypeptide comprising abinding site for an anti-PRO antibody or as antisense oligonucleotideprobes. Such nucleic acid fragments are usually at least about 20nucleotides in length, preferably at least about 30 nucleotides inlength, more preferably at least about 40 nucleotides in length, yetmore preferably at least about 50 nucleotides in length, yet morepreferably at least about 60 nucleotides in length, yet more preferablyat least about 70 nucleotides in length, yet more preferably at leastabout 80 nucleotides in length, yet more preferably at least about 90nucleotides in length, yet more preferably at least about 100nucleotides in length, yet more preferably at least about 110nucleotides in length, yet more preferably at least about 120nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleotides in length and yet more preferably at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PROpolypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the PRO polypeptide-encoding nucleotide sequence withother known nucleotide sequences using any of a number of well knownsequence alignment programs and determining which PROpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

[0260] In another embodiment, the invention provides isolated PROpolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0261] In a certain aspect, the invention concerns an isolated PROpolypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

[0262] In a further aspect, the invention concerns an isolated PROpolypeptide comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to an aminoacid sequence encoded by any of the human protein cDNAs deposited withthe ATCC as disclosed herein.

[0263] In a further aspect, the invention concerns an isolated PROpolypeptide comprising an amino acid sequence scoring at least about 80%positives, preferably at least about 81% positives, more preferably atleast about 82% positives, yet more preferably at least about 83%positives, yet more preferably at least about 84% positives, yet morepreferably at least about 85% positives, yet more preferably at leastabout 86% positives, yet more preferably at least about 87% positives,yet more preferably at least about 88% positives, yet more preferably atleast about 89% positives, yet more preferably at least about 90%positives, yet more preferably at least about 91% positives, yet morepreferably at least about 92% positives, yet more preferably at leastabout 93% positives, yet m ore preferably at least about 94% positives,yet more preferably at least about 95% positives, yet more preferably atleast about 96% positives, yet more preferably at least about 97%positives, yet more preferably at least about 98% positives and yet morepreferably at least about 99% positives when compared with the aminoacid sequence of a PRO polypeptide having a full-length amino acidsequence as disclosed herein, an amino acid sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a transmembraneprotein, with or without the signal peptide, as disclosed herein or anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein.

[0264] In a specific aspect, the invention provides an isolated PROpolypeptide without the N-terminal signal sequence and/or the initiatingmethionine and is encoded by a nucleotide sequence that encodes such anamino acid sequence as hereinbefore described. Processes for producingthe same are also herein described, wherein those processes compriseculturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the PRO polypeptide and recovering the PRO polypeptidefrom the cell culture.

[0265] Another aspect the invention provides an isolated PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

[0266] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

[0267] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists to a PRO polypeptide which comprisecontacting the PRO polypeptide with a candidate molecule and monitoringa biological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

[0268] In a still further embodiment, the invention concerns acomposition of matter comprising a PRO polypeptide, or an agonist orantagonist of a PRO polypeptide as herein described, or an anti-PROantibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

[0269] Another embodiment of the present invention is directed to theuse of a PRO polypeptide, or an agonist or antagonist thereof ashereinbefore described, or an anti-PRO antibody, for the preparation ofa medicament useful in the treatment of a condition which is responsiveto the PRO polypeptide, an agonist or antagonist thereof or an anti-PROantibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0270]FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a nativesequence PRO1800 cDNA, wherein SEQ ID NO:1 is a clone designated hereinas “DNA35672-2508”.

[0271]FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived fromthe coding sequence of SEQ ID NO:1 shown in FIG. 1.

[0272]FIG. 3 shows a nucleotide sequence (SEQ ID NO:6) of a nativesequence PRO539 cDNA, wherein SEQ ID NO:6 is a clone designated hereinas “DNA47465-1561”.

[0273]FIG. 4 shows the amino acid sequence (SEQ ID NO:7) derived fromthe coding sequence of SEQ ID NO:6 shown in FIG. 3.

[0274]FIG. 5 shows a nucleotide sequence (SEQ ID NO:8) of a nativesequence PRO982 cDNA, wherein SEQ ID NO:8 is a clone designated hereinas “DNA57700-1408”.

[0275]FIG. 6 shows the amino acid sequence (SEQ ID NO:9) derived fromthe coding sequence of SEQ ID NO:8 shown in FIG. 5.

[0276]FIG. 7 shows a nucleotide sequence (SEQ ID NO:12) of a nativesequence PRO1434 cDNA, wherein SEQ ID NO:12 is a clone designated hereinas “DNA68818-2536”.

[0277]FIG. 8 shows the amino acid sequence (SEQ ID NO:13) derived fromthe coding sequence of SEQ ID NO:12 shown in FIG. 7.

[0278]FIG. 9 shows a nucleotide sequence (SEQ ID NO:17) of a nativesequence PRO1863 cDNA, wherein SEQ ID NO:17 is a clone designated hereinas “DNA59847-2510”.

[0279]FIG. 10 shows the amino acid sequence (SEQ ID NO:18) derived fromthe coding sequence of SEQ ID NO:17 shown in FIG. 9.

[0280]FIG. 11 shows a nucleotide sequence (SEQ ID NO:19) of a nativesequence PRO1917cDNA, wherein SEQ ID NO:19 is a clone designated hereinas “DNA76400-2528”.

[0281]FIG. 12 shows the amino acid sequence (SEQ ID NO:20) derived fromthe coding sequence of SEQ ID NO:19 shown in FIG. 11.

[0282]FIG. 13 shows a nucleotide sequence (SEQ ID NO:21) of a nativesequence PRO1868 cDNA, wherein SEQ ID NO:21 is a clone designated hereinas “DNA77624-2515”.

[0283]FIG. 14 shows the amino acid sequence (SEQ ID NO:22) derived fromthe coding sequence of SEQ ID NO:21 shown in FIG. 13.

[0284]FIG. 15 shows a nucleotide sequence (SEQ ID NO:23) of a nativesequence PRO3434 cDNA, wherein SEQ ID NO:23 is a clone designated hereinas “DNA77631-2537”.

[0285]FIG. 16 shows the amino acid sequence (SEQ ID NO:24) derived fromthe coding sequence of SEQ ID NO:23 shown in FIG. 15.

[0286]FIG. 17 shows a nucleotide sequence (SEQ ID NO:25) of a nativesequence PRO1927 cDNA, wherein SEQ ID NO:25 is a clone designated hereinas “DNA82307-2531”.

[0287]FIG. 18 shows the amino acid sequence (SEQ ID NO:26) derived fromthe coding sequence of SEQ ID NO:25 shown in FIG. 17.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0288] I. Definitions

[0289] The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

[0290] A “native sequence PRO polypeptide” comprises a polypeptidehaving the same amino acid sequence as the corresponding PRO polypeptidederived from nature. Such native sequence PRO polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence PRO polypeptide” specificallyencompasses naturally-occurring truncated or secreted forms of thespecific PRO polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

[0291] The PRO polypeptide “extracellular domain” or “ECD” refers to aform of the PRO polypeptide which is essentially free of thetransmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECDwill have less than 1% of such transmembrane and/or cytoplasmic domainsand preferably, will have less than 0.5% of such domains. It will beunderstood that any transmembrane domains identified for the PROpolypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as initially identified herein. Optionally, therefore, anextracellular domain of a PRO polypeptide may contain from about 5 orfewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified in the Examples orspecification and such polypeptides, with or without the associatedsignal peptide, and nucleic acid encoding them, are comtemplated by thepresent invention.

[0292] The approximate location of the “signal peptides” of the variousPRO polypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. End. 10: 1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These polypeptides, wherethe signal peptide is cleaved within no more than about 5 amino acids oneither side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

[0293] “PRO polypeptide variant” means an active PRO polypeptide asdefined above or below having at least about 80% amino acid sequenceidentity with a full-length native sequence PRO polypeptide sequence asdisclosed herein, a PRO polypeptide sequence lacking the signal peptideas disclosed herein, an extracellular domain of a PRO polypeptide, withor without the signal peptide, as disclosed herein or any other fragmentof a full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, preferably at least about 81% amino acid sequence identity,more preferably at least about 82% amino acid sequence identity, morepreferably at least about 83% amino acid sequence identity, morepreferably at least about 84% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 86% amino acid sequence identity, morepreferably at least about 87% amino acid sequence identity, morepreferably at least about 88% amino acid sequence identity, morepreferably at least about 89% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 91% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, morepreferably at least about 93% amino acid sequence identity, morepreferably at least about 94% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 96% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, morepreferably at least about 98% amino acid sequence identity and mostpreferably at least about 99% amino acid sequence identity with afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, often at least about 20 amino acids inlength, more often at least about 30 amino acids in length, more oftenat least about 40 amino acids in length, more often at least about 50amino acids in length, more often at least about 60 amino acids inlength, more often at least about 70 amino acids in length, more oftenat least about 80 amino acids in length, more often at least about 90amino acids in length, more often at least about 100 amino acids inlength, more often at least about 150 amino acids in length, more oftenat least about 200 amino acids in length, more often at least about 300amino acids in length, or more.

[0294] “Percent (%) amino acid sequence identity” with respect to thePRO polypeptide sequences identified herein is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

[0295] In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0296] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program ALIGN-2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. As examples of % amino acid sequenceidentity calculations using this method, Tables 2 and 3 demonstrate howto calculate the % amino acid sequence identity of the amino acidsequence designated “Comparison Protein” to the amino acid sequencedesignated “PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

[0297] Unless specifically stated otherwise, all % amino acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

[0298] Percent amino acid sequence identity may also be determined usingthe sequence comparison program NCBI-BLAST2 (Altschul et al., NucleicAcids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparisonprogram may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2uses several search parameters, wherein all of those search parametersare set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

[0299] In situations where NCBI-BLAST2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0300] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

[0301] “PRO variant polynucleotide” or “PRO variant nucleic acidsequence” means a nucleic acid molecule which encodes an active PROpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleotide acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal peptide, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Ordinarily, a PRO variant polynucleotide will have atleast about 80% nucleic acid sequence identity, more preferably at leastabout 81% nucleic acid sequence identity, more preferably at least about82% nucleic acid sequence, identity, more preferably at least about 83%nucleic acid sequence identity, more preferably at least about 84%nucleic acid sequence identity, more preferably at least about 85%nucleic acid sequence identity, more preferably at least about 86%nucleic acid sequence identity, more preferably at least about 87%nucleic acid sequence identity, more preferably at least about 88%nucleic acid sequence identity, more preferably at least about 89%nucleic acid sequence identity, more preferably at least about 90%nucleic acid sequence identity, more preferably at least about 91%nucleic acid sequence identity, more preferably at least about 92%nucleic acid sequence identity, more preferably at least about 93%nucleic acid sequence identity, more preferably at least about 94%nucleic acid sequence identity, more preferably at least about 95%nucleic acid sequence identity, more preferably at least about 96%nucleic acid sequence identity, more preferably at least about 97%nucleic acid sequence identity, more preferably at least about 98%nucleic acid sequence identity and yet more preferably at least about99% nucleic acid sequence identity wi th a nucleic acid sequenceencoding a full-length native sequence PRO polypeptide sequence asdisclosed herein, a full-length native sequence PRO polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a PRO polypeptide, with or without the signal sequence, as disclosedherein or any other fragment of a full-length PRO polypeptide sequenceas disclosed herein. Variants do not encompass the native nucleotidesequence.

[0302] Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, often at least about 60 nucleotides in length,more often at least about 90 nucleotides in length, more often at leastabout 120 nucleotides in length, more often at least about 150nucleotides in length, more often at least about 180 nucleotides inlength, more often at least about 210 nucleotides in length, more oftenat least about 240 nucleotides in length, more often at least about 270nucleotides in length, more often at least about 300 nucleotides inlength, more often at least about 450 nucleotides in length, more oftenat least about 600 nucleotides in length, more often at least about 900nucleotides in length, or more.

[0303] “Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

[0304] In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

[0305] where W is the number of nucleotides scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

[0306] Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

[0307] Percent nucleic acid sequence identity may also be determinedusing the sequence comparison program NCBI-BLAST2 (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequencecomparison program may be downloaded from http://www.ncbi.nlm.nih.gov.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment 25 and scoring matrix=BLOSUM62.

[0308] In situations where NCBI-BLAST2 is employed for sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

[0309] where W is the number of nucleotides scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of C and D, and where Z is the total number of nucleotides inD. It will be appreciated that where the length of nucleic acid sequenceC is not equal to the length of nucleic acid sequence D, the % nucleicacid sequence identity of C to D will not equal the % nucleic acidsequence identity of D to C.

[0310] In other embodiments, PRO variant polynucleotides are nucleicacid molecules that encode an active PRO polypeptide and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

[0311] The term “positives”, in the context of sequence comparisonperformed as described above, includes residues in the sequencescompared that are not identical but have similar properties (e.g. as aresult of conservative substitutions, see Table 6 below). For purposesherein, the % value of positives is determined by dividing (a) thenumber of amino acid residues scoring a positive value between the PROpolypeptide amino acid sequence of interest having a sequence derivedfrom the native PRO polypeptide sequence and the comparison amino acidsequence of interest (i.e., the amino acid sequence against which thePRO polypeptide sequence is being compared) as determined in theBLOSUM62 matrix of WU-BLAST-2 by (b) the total number of amino acidresidues of the PRO polypeptide of interest.

[0312] Unless specifically stated otherwise, the % value of positives iscalculated as described in the immediately preceding paragraph. However,in the context of the amino acid sequence identity comparisons performedas described for ALIGN-2 and NCBI-BLAST-2 above, includes amino acidresidues in the sequences compared that are not only identical, but alsothose that have similar properties. Amino acid residues that score apositive value to an amino acid residue of interest are those that areeither identical to the amino acid residue of interest or are apreferred substitution (as defined in Table 6 below) of the amino acidresidue of interest.

[0313] For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2,the % value of positives of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% positives to, with, or against a given amino acid sequence B) iscalculated as follows:

100 times the fraction X/Y

[0314] where X is the number of amino acid residues scoring a positivevalue as defined above by the sequence alignment program ALIGN-2 orNCBI-BLAST2 in that program's alignment of A and B, and where Y is thetotal number of amino acid residues in B. It will be appreciated thatwhere the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % positives of A to B will not equal the %positives of B to A.

[0315] “Isolated,” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

[0316] An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

[0317] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0318] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0319] The term “antibody” is used in the broadest sense andspecifically covers, for example, single anti-PRO monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies), anti-PROantibody compositions with polyepitopic specificity, single chainanti-PRO antibodies, and fragments of anti-PRO antibodies (see below).The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts.

[0320] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0321] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0322] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and%SDS) less stringent that those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

[0323] The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

[0324] As used herein, the term “immunoadhesins” designatesantibody-like molecules which combine the binding specificity of aheterologous protein (an “adhesin”) with the effector functions ofimmunoglobulin constant domains. Structurally, the immunoadhesinscomprise a fusion of an amino acid sequence with the desired bindingspecificity which is other than the antigen recognition and binding siteof an antibody (i.e., is “heterologous”), and an immunoglobulin constantdomain sequence. The adhesin part of an immunoadhesin molecule typicallyis a contiguous amino acid sequence comprising at least the binding siteof a receptor or a ligand. The immunoglobulin constant domain sequencein the immunoadhesin may be obtained from any immunoglobulin, such asIgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2),IgE, IgD or IgM.

[0325] “Active” or “activity” for the purposes herein refers to form(s)of a PRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

[0326] The term “antagonist” is used in the broadest sense, and includesany molecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

[0327] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

[0328] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

[0329] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0330] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0331] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0332] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0333] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. Pepsin treatment yieldsan F(ab′)₂ fragment that has two antigen-combining sites and is stillcapable of cross-linking antigen.

[0334] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0335] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fabfragments differ from Fab′ fragments by the addition of a few residuesat the carboxy terminus of the heavy chain CH1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

[0336] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains.

[0337] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0338] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

[0339] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0340] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0341] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.

[0342] By “solid phase” is meant a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

[0343] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

[0344] A “small molecule” is defined herein to have a molecular weightbelow about 500 Daltons. TABLE 1 /*  *  * C-C increased from 12 to 15  *Z is average of EQ  * B is average of ND  * match with stop is _M;stop-stop = 0; J (joker) match = 0  */ #define _M −8 /* value of a matchwith a stop */ int _day[26][26] = { /*  A B C D E F G H I J K L M N O PQ R S T U V W X Y Z */ /* A */ {2, 0, −2, 0, 0, −4, 1, −1, −1, 0, −1,−2, −1, 0, _M, 1, 0, −2, 1, 1, 0, 0, −6, 0, −3, 0}, /* B */ {0, 3, −4,3, 2, −5, 0, 1, −2, 0, 0, −3, −2, 2, _M, −1, 1, 0, 0, 0, 0, −2, −5, 0,−3, 1}, /* C */ {−2, −4, 15, −5, −5, −4, −3, −3, −2, 0, −5, −6, −5, −4,_M, −3, −5, −4, 0, −2, 0, −2, −8, 0, 0, −5}, /* D */ {0, 3, −5, 4, 3,−6, 1, 1, −2, 0, 0, −4, −3, 2, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4,2}, /* E */ {0, 2, −5, 3, 4, −5, 0, 1, −2, 0, 0, −3, −2, 1, _M, −1, 2,−1, 0, 0, 0, −2, −7, 0, −4, 3}, /* F */ {−4, −5, −4, −6, −5, 9, −5, −2,1, 0, −5, 2, 0, −4, _M, −5, −5, −4, −3, −3, 0, −1, 0, 0, 7, −5}, /* G */{1, 0, −3, 1, 0, −5, 5, −2, −3, 0, −2, −4, −3, 0, _M, −1, −1, −3, 1, 0,0, −1, −7, 0, −5, 0}, /* H */ {−1, 1, −3, 1, 1, −2, −2, 6, −2, 0, 0, −2,−2, 2, _M, 0, 3, 2, −1, −1, 0, −2, −3, 0, 0, 2}, /* I */ {−1, −2, −2,−2, −2, 1, −3, −2, 5, 0, −2, 2, 2, −2, _M, −2, −2, −2, −1, 0, 0, 4, −5,0, −1, −2}, /* J */ {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */ {−1, 0, −5, 0, 0, −5, −2, 0, −2, 0,5, −3, 0, 1, _M, −1, 1, 3, 0, 0, 0, −2, −3, 0, −4, 0}, /* L */ {−2, −3,−6, −4, −3, 2, −4, −2, 2, 0, −3, 6, 4, −3, _M, −3, −2, −3, −3 , −1, 0,2, −2, 0, −1, −2} /* M */ {−1, −2, −5, −3, −2, 0, −3, −2, 2, 0, 0, 4, 6,−2, _M, −2, −1, 0, −2, −1, 0, 2, −4, 0, −2, −1}, /* N */ {0, 2, −4, 2,1, −4, 0, 2, −2, 0, 1, −3, −2, 2, _M, −1, 1, 0, 1, 0, 0, −2, −4, 0, −2,1}, /* O */ {_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,}, /* P */ {1, −1, −3, −1, −1, −5,−1, 0, −2, 0, −1, −3, −2, −1,_M, 6, 0, 0, 1, 0, 0, −1, −6, 0, −5, 0}, /*Q */ {0, 1, −5, 2, 2, −5, −1, 3, −2, 0, 1, −2, −1, 1, _M, 0, 4, 1, −1,−1, 0, −2, −5, 0, −4, 3}, /* R */ {−2, 0, −4, −1, −1, −4, −3, 2, −2, 0,3, −3, 0, 0, _M, 0, 1, 6, 0, −1, 0, −2, 2, 0, −4, 0}, /* S */ {1, 0, 0,0, 0, −3, 1, −1, −1, 0, 0, −3, −2, 1, _M, 1, −1, 0, 2, 1, 0, −1, −2, 0,−3, 0}, /* T */ {1, 0, −2, 0, 0, −3, 0, −1, 0, 0, 0, −1, −1, 0, _M, 0,−1, −1, 1, 3, 0, 0, −5, 0, −3, 0}, /* U */ {0, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {0, −2, −2,−2, −2, −1, −1, −2, 4, 0, −2, 2, 2, −2,_M, −1, −2, −2, −1, 0, 0, 4, −6,0, −2, −2}, /* W */ {−6, −5, −8, −7, −7, 0, −7, −3, −5, 0, −3, −2, −4,−4,_M, −6, −5, 2, −2, −5, 0, −6, 17, 0, 0, −6}, /* X */ {0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */{−3, −3, 0, −4, −4, 7, −5, 0, −1, 0, −4, −1, −2, −2, _M, −5, −4, −4, −3,−3, 0, −2, 0, 0, 10, −4}, /* Z */ {0, 1, −5, 2, 3, −5, 0, 2, −2, 0, 0,−2, −1, 1,_M, 0, 3, 0, 0, 0, 0, −2, −6, 0, −4, 4}, }; /*  */ #include<stdio.h> #include <ctype.h> #define MAXJMP  16 /* max jumps in a diag*/ #define MAXGAP  24 /* don't continue to penalize gaps larger thanthis */ #define JMPS 1024 /* max jmps in an path */ #define MX   4 /*save if there's at least MX-1 bases since last jmp */ #define DMAT   3/* value of matching bases */ #define DMIS   0 /* penalty for mismatchedbases */ #define DINS0   8 /* penalty for a gap */ #define DINS1   1 /*penalty per base */ #define PINS0   8 /* penalty for a gap */ #definePINS1   4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /*size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. ofjmp in seq x */ /* limits seq to 2{circumflex over ( )}16 −1 */ };struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for errmsgs */ char *seqx[2];   /* seqs: getseqs() */ int dmax; /* best diag:nw() */ int dmax0; /* final diag */ int dna; /* set if dna: main() */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw() */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char*getseq(), *g_calloc(); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  * where file1 and file2 are two dna or twoprotein sequences.  * The sequences can be in upper- or lower-case anmay contain ambiguity  * Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  * Max file length is 65535 (limited by unsigned short x in thejmp struct)  * A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  * Output is in the file “align.out”  *  * The programmay create a tmp file in /tmp to hold info about traceback.  * Originalversion developed under BSD 4.3 on a vax 8650  */ #include “nw.h”#include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1< <(‘D’-‘A’))|(1< <(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0×FFFFFFF, 1< <10, 1< <11, 1< <12, 1< <13, 1< <14, 1< <15, 1<<16, 1< <17, 1< <18, 1< <19, 1< <20, 1< <21, 1< <22, 1< <23, 1< <24, 1<<25|(1< <(‘E’-‘A’))|(1< <(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[]; { prog = av[0]; if(ac != 3) { fprintf(stderr, “usage: %s file1file2\n”, prog); fprintf(stderr, “where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr, “The sequences can be inupper- or lower-case\n”); fprintf(stderr, “Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr, “Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw(); /* fill in the matrix, getthe possible jmps */ readjmps(); /* get the actual jmps */ print(); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main()  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw() nw { char *px, *py;   /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PlNS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy= 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0]−ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx = 0;} ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis + = (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if(col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] =−ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy);if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps &&xx < len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) {smax = col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }(void) free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print() -- only routinevisible outside this module  *  * static:  * getmat() -- trace back bestpath, count matches: print()  * pr_align() -- print alignment ofdescribed in array p[]: print()  * dumpblock() -- dump a block of lineswith numbers, stars: pr_align()  * nums() -- put out a number line:dumpblock()  * putline() -- put out a line (name, [num], seq, [num]):dumpblock()  * stars() - -put a line of stars: dumpblock()  *stripname() -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC  3 #define P_LINE 256 /* maximum output line */#define P_SPC  3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print() print { int lx, ly, firstgap, lastgap;  /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr, “%s: can't write%s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1;firstgap = lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align(); } /*  * traceback the best path, count matches  */ static getmat(lx, ly, firstgap,lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap,lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1;char outx[32]; double pct; register n0, n1; register char *p0, *p1; /*get total matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (nl++ == pp[1].x[i1]) siz1 = pp[1].n[il++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “(%d %s%s)”, ngapx, (dna)?“base”: “residue”, (ngapx == 1)? “”:“s”); fprintf(fx, “% s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “(%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy == 1)?“”:“s”); fprintf(fx, “%s”, outx); } if (dna) fprintf(fx, “\n<score: %d(match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars() */ /*  * print alignment of described in struct path pp[]  */static pr_align() pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2++) { nn = stripname(namex[i]); if (nn >lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more;) {...pr_align for (i = more = 0; i < 2; i++) { /*  * do we have more ofthis sequence?  */ if (!*ps[i]) continue; more++; if (pp[i].spc) { /*leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if (siz[i]) { /* ina gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we're putting a seqelement */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] == pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock(); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align()  */ static dumpblock() dumpblock { register i; for(i =0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx); for(i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) != ‘’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars(); putline(i);if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } } *put out a number line: dumpblock()  */ static nums(ix) nums int  ix; /*index in out[] holding seq line */ { char nline[P_LINE]; register i, j;register char *pn, *px, *py; for(pn = nline, i = 0; i < lmax+P_SPC; i++,pn++) *pn = ‘ ’; for (i = nc[ix], py = out[ix]; *py; py++, pn++) { if(*py == ‘ ’ || *py == ‘−’); *pn = ‘ ’; else { if (i%10 == 0 || (i == 1&& nc[ix] != 1)) { j = (i < 0)? −i : i; for (px = pn; j; j/= 10, px−−)*px = j%10 + ‘0’; if (i < 0) *px = ‘−’; } else *pn = ‘ ’; i++; } } *pn =‘\0’; nc[ix] = i; for (pn = nline; *pn; pn++) (void) putc(*pn, fx);(void) putc(‘\n’, fx); } /*  * put out a line (name, [num], seq. [num]):dumpblock()  */ static putline(ix) putline int   ix; { ...putline int i;register char *px; for (px = namex[ix], i = 0; *px && *px != ‘:’; px++,i++) (void) putc(*px, fx); for (;i < lmax+P_SPC; i++) (void) putc(‘ ’,fx); /* these count from 1:  * ni[] is current element (from 1)  * nc[]is number at start of current line  */ for (px = out[ix]; *px; px++)(void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx); } /*  * put a line ofstars (seqs always in out[0], out[1]): dumpblock()  */ static stars()stars { int i; register char *p0, *p1, cx, *px; if (!*out[0] || (*out[0]== ‘ ’ && *(p0[0]) == ‘ ’) || !*out[1] || (*out[1] == ‘ ’ && *(po[1]) ==‘ ’)) return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for(p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0− ‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align()  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup() --cleanup any tmp file  * getseq() -- read in seq, set dna, len, maxlen  *g_calloc() -- calloc() with error checkin  * readjmps() -- get the goodjmps, from tmp file if necessary  * writejmps() -- write a filled arrayof jmps to a tmp file: nw()  */ #include “nw.h” #include <sys/file.h>char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */ FILE *fj; intcleanup(); /* cleanup tmp file */ long lseek(); /*  * remove any tmpfile if we blow  */ cleanup(i) cleanup int i; { if (fj) (void)unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna, len,maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upper orlower case  */ char * getseq(file, len) getseq char *file; /* file name*/ int *len; /* seq len */ { char line[1024], *pseq; register char *px,*py; int natgc, tlen; FILE *fp; if ((fp = fopen(file, “r”)) == 0) {fprintf(stderr, “%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr, “%s: malloc() failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ||*line == ‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”, *(py−1))) natgc++; } } *py++ = ‘\0’;*py = ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc() failed %s (n= %d, sz= %d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[] or tmp file, set pp[], reset dmax: main()  */ readjmps() readjmps {int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) { (void)fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr,“%s: can't open() %s\n”, prog, jname); cleanup(1); } } for (i = i0 = i1= 0, dmax0 = dmax, xx = len0; ;i++) { while (1) { for (j =dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmps if(j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset, 0);(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[il] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[il] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; il++; } else if (siz > 0) { /* gapin first seq */ pp[0] .n[i0] = siz; pp[0] .x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw()  */writejmps(ix) writejmps int ix; { char *mktemp(); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp() %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

[0345] TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

[0346] TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

[0347] TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acidsequence identity = (the number of identically matching nucleotidesbetween the two nucleic acid sequences as determined by ALIGN-2) dividedby (the total number of nucleotides of the PRO-DNA nucleic acidsequence) = 6 divided by 14 = 42.9%

[0348] TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonNNNNLLLVV (Length = 9 nucleotides) DNA % nucleic acid sequence identity= (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the PRO-DNA nucleic acid sequence) = 4 dividedby 12 = 33.3%

[0349] II. Compositions and Methods of the Invention

[0350] A. Full-Length PRO Polypeptides

[0351] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO polypeptides. In particular, cDNAs encoding variousPRO polypeptides have been identified and isolated, as disclosed infurther detail in the Examples below. It is noted that proteins producedin separate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

[0352] As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

[0353] 1. Full-length PRO1800 Polypeptides

[0354] Using the WU-BLAST2 sequence alignment computer program, it hasbeen found that a portion of the full-length native sequence PRO1800(shown in FIG. 2 and SEQ ID NO:2) has certain amino acid sequenceidentity with the human Hep27 protein (HE27_HUMAN). Accordingly, it ispresently believed that PRO1800 disclosed in the present application isa newly identified Hep27 homolog and possesses activity typical of thatprotein.

[0355] 2. Full-length PRO539 Polypeptides

[0356] Using the WU-BLAST2 sequence alignment computer program, it hasbeen found that a portion of the full-length native sequence PRO539(shown in FIG. 4 and SEQ ID NO:7) has certain amino acid sequenceidentity with a portion of a kinesin-related protein from Drosophilamelanogaster (AF019250_(—)1). Accordingly, it is presently believed thatPRO539 disclosed in the present application is a newly identified memberof the Hedgehog signaling pathway protein family and possesses activitytypical of the Drosophila Costal-2 protein.

[0357] 3. Full-length PRO982 Polypeptides

[0358] As far as is known, the DNA57700-1408 sequence encodes a novelsecreted factor designated herein as PRO982. Although, using WU-BLAST2sequence alignment computer programs, some sequence identities withknown proteins were revealed.

[0359] 4. Full-length PRO1434 Polypeptides

[0360] Using the WU-BLAST2 sequence alignment computer program, it hasbeen found that a portion of the full-length native sequence PRO1434(shown in FIG. 10 and SEQ ID NO:13) has certain amino acid sequenceidentity with the mouse nel protein precursor (NEL_MOUSE). Accordingly,it is presently believed that PRO1434 disclosed in the presentapplication is a newly identified nel homolog and may possess activitytypical of the nel protein family.

[0361] 5. Full-length PRO1863 Polypeptides

[0362] The DNA59847-2510 clone was isolated from a human prostate tissuelibrary. As far as is known, the DNA59847-2510 sequence encodes a novelfactor designated herein as PRO1863; using the WU-BLAST2 sequencealignment computer program, no significant sequence identities to anyknown proteins were revealed.

[0363] 6. Full-length PRO1917 Polypeptides

[0364] Using WU-BLAST2 sequence alignment computer programs, it has beenfound that amino acids 41 to 487 of PRO1917 (shown in FIG. 14 and SEQ IDNO:20) has certain amino acid sequence identity with an inositolphosphatase designated in the Dayhoff database as “AF012714_(—)1”.Accordingly, it is presently believed that PRO1917 disclosed in thepresent application is a newly identified member of inositol phosphatasefamily and may possess enzymatic activity typical of inositolphosphatases.

[0365] 7. Full-length PRO1868 Polypeptides

[0366] Using the WU-BLAST2 sequence alignment computer program, it hasbeen found that a portion of the full-length native sequence PRO1868(shown in FIG. 16 and SEQ ID NO:28) has certain amino acid sequenceidentity with the human A33 antigen protein (P_W14146). Accordingly, itis presently believed that PRO1868 disclosed in the present applicationis a newly identified A33 antigen homolog which may possess activityand/or expression patterns typical of the A33 antigen protein. ThePRO1868 polypeptide may find use in the therapeutic treatment ofinflammatory diseases as described above and colorectal cancer.

[0367] 8. Full-length PRO3434 Polypeptides

[0368] The DNA77631-2537 clone was isolated from a human aortic tissuelibrary using a trapping technique that selects for nucleotide sequencesencoding secreted proteins. As far as is known, the DNA77631-2537sequence encodes a novel factor designated herein as PRO3434; using theWU-BLAST2 sequence alignment computer program, no significant sequenceidentities to any known proteins were revealed.

[0369] 9. Full-length PRO1927 Polypeptides

[0370] Using WU-BLAST2 sequence alignment computer programs, it has beenfound that a full-length native sequence PRO1927 (FIG. 20; SEQ ID NO:26)has certain amino acid sequence identity with the amino acid sequence ofthe protein designated “AB000628_(—)1” in the Dayhoff database.Accordingly, it is presently believed that PRO1927 disclosed in thepresent application is a newly identified member of theglycosyltransferase family of proteins and may possess glycosylationactivity.

[0371] B. PRO Polypeptide Variants

[0372] In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

[0373] Variations in the native full-length sequence PRO or in variousdomains of the PRO described herein, can be made, for example, using anyof the techniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

[0374] PRO polypeptide fragments are provided herein. Such fragments maybe truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

[0375] PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

[0376] In particular embodiments, conservative substitutions of interestare shown in Table 1 under the heading of preferred substitutions. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 6 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucineleu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln;asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leuPro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr(Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala;norleucine

[0377] Substantial modifications in function or immunologicalidentity ofthe PRO polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0378] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0379] (2) neutral hydrophilic: cys, ser, thr;

[0380] (3) acidic: asp, glu;

[0381] (4) basic: asn, gln, his, lys, arg;

[0382] (5) residues that influence chain orientation: gly, pro; and

[0383] (6) aromatic: trp, tyr, phe.

[0384] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class. Such substituted residues alsomay be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

[0385] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

[0386] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

[0387] C. Modifications of PRO

[0388] Covalent modifications of PRO are included within the scope ofthis invention. One type of covalent modification includes reactingtargeted amino acid residues of a PRO polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the PRO. Derivatization withbifunctional agents is useful, for instance, for crosslinking PRO to awater-insoluble support matrix or surface for use in the method forpurifying anti-PRO antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0389] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0390] Another type of covalent modification of the PRO polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

[0391] Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

[0392] Another means of increasing the number of carbohydrate moietieson the PRO polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0393] Removal of carbohydrate moieties present on the PRO polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

[0394] Another type of covalent modification of PRO comprises linkingthe PRO polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0395] The PRO of the present invention may also be modified in a way toform a chimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

[0396] In one embodiment, such a chimeric molecule comprises a fusion ofthe PRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

[0397] In an alternative embodiment, the chimeric molecule may comprisea fusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

[0398] D. Preparation of PRO

[0399] The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W. H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

[0400] 1. Isolation of DNA Encoding PRO

[0401] DNA encoding PRO may be obtained from a cDNA library preparedfrom tissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

[0402] Libraries can be screened with probes (such as antibodies to thePRO or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding PRO is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

[0403] The Examples below describe techniques for screening a cDNAlibrary. The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The oligonucleotide is preferably labeled such that it can bedetected upon hybridization to DNA in the library being screened.Methods of labeling are well known in the art, and include the use ofradiolabels like ³²P-labeled ATP, biotinylation or enzyme labeling.Hybridization conditions, including moderate stringency and highstringency, are provided in Sambrook et al., supra.

[0404] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined using methods known in the art and as described herein.

[0405] Nucleic acid having protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

[0406] 2. Selection and Transformation of Host Cells

[0407] Host cells are transfected or transformed with expression orcloning vectors described herein for PRO production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

[0408] Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

[0409] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Eschenichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan ^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

[0410] In addition to prokaryotes, eukaryotic microbes such asfilamentous fumgi or yeast are suitable cloning or expression hosts forPRO-encoding vectors. Saccharomyces cerevisiae is a commonly used lowereukaryotic host microorganism. Others include Schizosaccharomyces pombe(Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published May 2,1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastonis(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

[0411] Suitable host cells for the expression of glycosylated PRO arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

[0412] 3. Selection and Use of a Replicable Vector

[0413] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

[0414] The PRO may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

[0415] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells.

[0416] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0417] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

[0418] Expression and cloning vectors usually contain a promoteroperably linked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

[0419] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvatedecarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphateisomerase, phosphoglucose isomerase, andglucokinase.

[0420] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0421] PRO transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and Simian Virus 40 (SV40), from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, and fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

[0422] Transcription of a DNA encoding the PRO by higher eukaryotes maybe increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

[0423] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding PRO.

[0424] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of PRO in recombinant vertebrate cellculture are described in Gething et al., Nature, 293:620-625 (1981);Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

[0425] 4. Detecting Gene Amplification/Expression

[0426] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0427] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

[0428] 5. Purification of Polypeptide

[0429] Forms of PRO may be recovered from culture medium or from hostcell lysates. If membrane-bound, it can be released from the membraneusing a suitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

[0430] It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

[0431] E. Uses for PRO

[0432] Nucleotide sequences (or their complement) encoding PRO havevarious applications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

[0433] The full-length native sequence PRO gene, or portions thereof,may be used as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

[0434] Any EST sequences disclosed in the present application maysimilarly be employed as probes, using the methods disclosed herein.

[0435] Other useful fragments of the PRO nucleic acids include antisenseor sense oligonucleotides comprising a singe-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target PRO mRNA(sense) or PRO DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of PRO DNA. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 to 30nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

[0436] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that blocktranscription or translation of the target sequence by one of severalmeans, including enhanced degradation of the duplexes, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression of PROproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO 91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

[0437] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10048, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0438] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

[0439] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0440] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0441] Antisense or sense RNA or DNA molecules are generally at leastabout 5 bases in length, about 10 bases in length, about 15 bases inlength, about 20 bases in length, about 25 bases in length, about 30bases in length, about 35 bases in length, about 40 bases in length,about 45 bases in length, about 50 bases in length, about 55 bases inlength, about 60 bases in length, about 65 bases in length, about 70bases in length, about 75 bases in length, about 80 bases in length,about 85 bases in length, about 90 bases in length, about 95 bases inlength, about 100 bases in length, or more.

[0442] The probes may also be employed in PCR techniques to generate apool of sequences for identification of closely related PRO codingsequences.

[0443] Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

[0444] When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

[0445] Nucleic acids which encode PRO or its modified forms can also beused to generate either transgenic animals or “knock out” animals which,in turn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

[0446] Alternatively, non-human homologues of PRO can be used toconstruct a PRO “knock out” animal which has a defective or altered geneencoding PRO as a result of homologous recombination between theendogenous gene encoding PRO and altered genomic DNA encoding PROintroduced into an embryonic stem cell of the animal. For example, cDNAencoding PRO can be used to clone genomic DNA encoding PRO in accordancewith established techniques. A portion of the genomic DNA encoding PROcan be deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the PRO polypeptide.

[0447] Nucleic acid encoding the PRO polypeptides may also be used ingene therapy. In gene therapy applications, genes are introduced intocells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacement of a defectivegene. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

[0448] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al., Trends inBiotechnology 11, 205-210 [1993]). In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells, such as an antibody specific for a cell surface membrane proteinor the target cell, a ligand for a receptor on the target cell, etc.Where liposomes are employed, proteins which bind to a cell surfacemembrane protein associated with endocytosis may be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

[0449] The PRO polypeptides described herein may also be employed asmolecular weight markers for protein electrophoresis purposes and theisolated nucleic acid sequences may be used for recombinantly expressingthose markers.

[0450] The nucleic acid molecules encoding the PRO polypeptides orfragments thereof described herein are useful for chromosomeidentification. In this regard, there exists an ongoing need to identifynew chromosome markers, since relatively few chromosome markingreagents, based upon actual sequence data are presently available. EachPRO nucleic acid molecule of the present invention can be used as achromosome marker.

[0451] The PRO polypeptides and nucleic acid molecules of the presentinvention may also be used for tissue typing, wherein the PROpolypeptides of the present invention may be differentially expressed inone tissue as compared to another. PRO nucleic acid molecules will finduse for generating probes for PCR, Northern analysis, Southern analysisand Western analysis.

[0452] The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Oso1, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

[0453] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution.

[0454] Therapeutic compositions herein generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0455] The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

[0456] Dosages and desired drug concentrations of pharmaceuticalcompositions of the present invention may vary depending on theparticular use envisioned. The determination of the appropriate dosageor route of administration is well within the skill of an ordinaryphysician. Animal experiments provide reliable guidance for thedetermination of effective doses for human therapy. Interspecies scalingof effective doses can be performed following the principles laid downby Mordenti, J. and Chappell, W. “The use of interspecies scaling intoxicokinetics” In Toxicokinetics and New Drug Development, Yacobi etal., Eds., Pergamon Press, New York 1989, pp. 42-96.

[0457] When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

[0458] Where sustained-release administration of a PRO polypeptide isdesired in a formulation with release characteristics suitable for thetreatment of any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN−), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology, 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

[0459] The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

[0460] This invention encompasses methods of screening compounds toidentify those that mimic the PRO polypeptide (agonists) or prevent theeffect of the PRO polypeptide (antagonists). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the PRO polypeptides encoded by the genes identifiedherein, or otherwise interfere with the interaction of the encodedpolypeptides with other cellular proteins. Such screening assays willinclude assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

[0461] The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

[0462] All assays for antagonists are common in that they call forcontacting the drug candidate with a PRO polypeptide encoded by anucleic acid identified herein under conditions and for a timesufficient to allow these two components to interact.

[0463] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the PRO polypeptide encoded by the geneidentified herein or the drug candidate is immobilized on a solid phase,e.g., on a microtiter plate, by covalent or non-covalent attachments.Non-covalent attachment generally is accomplished by coating the solidsurface with a solution of the PRO polypeptide and drying.Alternatively, an immobilized antibody, e.g., a monoclonal antibody,specific for the PRO polypeptide to be immobilized can be used to anchorit to a solid surface. The assay is performed by adding thenon-immobilized component, which may be labeled by a detectable label,to the immobilized component, e.g., the coated surface containing theanchored component. When the reaction is complete, the non-reactedcomponents are removed, e.g., by washing, and complexes anchored on thesolid surface are detected. When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilizedon the surface indicates that complexing occurred. Where the originallynon-immobilized component does not carry a label, complexing can bedetected, for example, by using a labeled antibody specifically bindingthe immobilized complex.

[0464] If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

[0465] Compounds that interfere with the interaction of a gene encodinga PRO polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

[0466] To assay for antagonists, the PRO polypeptide may be added to acell along with the compound to be screened for a particular activityand the ability of the compound to inhibit the activity of interest inthe presence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

[0467] As an alternative approach for receptor identification, labeledPRO polypeptide can be photoaffinity-linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE and exposed to X-ray film. The labeledcomplex containing the receptor can be excised, resolved into peptidefragments, and subjected to protein micro-sequencing. The amino acidsequence obtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

[0468] In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

[0469] More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

[0470] Another potential PRO polypeptide antagonist is an antisense RNAor DNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Olieodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotidesderived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

[0471] Potential antagonists include small molecules that bind to theactive site, the receptor binding site, or growth factor or otherrelevant binding site of the PRO polypeptide, thereby blocking thenormal biological activity of the PRO polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

[0472] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques. For furtherdetails see, e.g., Rossi, Current Biology, 4:469-471 (1994), and PCTpublication No. WO 97/33551 (published Sep. 18, 1997).

[0473] Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

[0474] These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

[0475] F. Anti-PRO Antibodies

[0476] The present invention further provides anti-PRO antibodies.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

[0477] 1. Polyclonal Antibodies

[0478] The anti-PRO antibodies may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the PRO polypeptide or afusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0479] 2. Monoclonal Antibodies

[0480] The anti-PRO antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

[0481] The immunizing agent will typically include the PRO polypeptideor a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell [Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103]. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0482] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001(1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0483] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst PRO. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0484] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0485] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0486] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0487] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0488] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0489] 3. Human and Humanized Antibodies

[0490] The anti-PRO antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)].

[0491] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0492] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0493] 4. Bispecific Antibodies

[0494] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the PRO, the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

[0495] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0496] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0497] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0498] Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniquesfor generating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

[0499] Fab′ fragments may be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

[0500] Various technique for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994). Antibodieswith more than two valencies are contemplated. For example, trispecificantibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0501] Exemplary bispecific antibodies may bind to two differentepitopes on a given PRO polypeptide herein. Alternatively, an anti-PROpolypeptide arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular PRO polypeptide.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express a particular PRO polypeptide. These antibodiespossess a PRO-binding arm and an arm which binds a cytotoxic agent or aradionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Anotherbispecific antibody of interest binds the PRO polypeptide and furtherbinds tissue factor (TF).

[0502] 5. Heteroconjugate Antibodies

[0503] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0504] 6. Effector Function Engineering

[0505] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance, e.g., the effectivenessof the antibody in treating cancer. For example, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design. 3: 219-230 (1989).

[0506] 7. Immunoconjugates

[0507] The invention also pertains to immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate).

[0508] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleutites fordii proteins, dianthin proteins, Phytolacaameticana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

[0509] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as his(p-azidobenzoyl) hexanediamine), his-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

[0510] In another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0511] 8. Immunoliposomes

[0512] The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0513] Particularly useful liposomes can be generated by thereverse-phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, andPEG-derivatizedphosphatidylethanolamine(PEG-PE). Liposomes are extrudedthrough filters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

[0514] 9. Pharmaceutical Compositions of Antibodies

[0515] Antibodies specifically binding a PRO polypeptide identifiedherein, as well as other molecules identified by the screening assaysdisclosed hereinbefore, can be administered for the treatment of variousdisorders in the form of pharmaceutical compositions.

[0516] If the PRO polypeptide is intracellular and whole antibodies areused as inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

[0517] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

[0518] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0519] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S-S bond formation through thio-disulfideinterchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0520] G. Uses for Anti-PRO Antibodies

[0521] The anti-PRO antibodies of the invention have various utilities.For example, anti-PRO antibodies may be used in diagnostic assays forPRO, e.g., detecting its expression in specific cells, tissues, orserum. Various diagnostic assay techniques known in the art may be used,such as competitive binding assays, direct or indirect sandwich assaysand immunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

[0522] Anti-PRO antibodies also are useful for the affinity purificationof PRO from recombinant cell culture or natural sources. In thisprocess, the antibodies against PRO are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art. The immobilized antibody then is contacted with a samplecontaining the PRO to be purified, and thereafter the support is washedwith a suitable solvent that will remove substantially all the materialin the sample except the PRO, which is bound to the immobilizedantibody. Finally, the support is washed with another suitable solventthat will release the PRO from the antibody.

[0523] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0524] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0525] Commercially available reagents referred to in the examples wereused according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Manassas, Va.

Example 1

[0526] Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

[0527] The extracellular domain (ECD) sequences (including the secretionsignal sequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons with a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

[0528] Using this extracellular domain homology screen, consensus DNAsequences were assembled relative to the other identified EST sequencesusing phrap. In addition, the consensus DNA sequences obtained wereoften (but not always) extended using repeated cycles of BLAST orBLAST-2 and phrap to extend the consensus sequence as far as possibleusing the sources of EST sequences discussed above.

[0529] Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

[0530] The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

Example 2

[0531] Isolation of cDNA Clones by Amylase Screening

[0532] 1. Preparation of oligo dT primed cDNA library

[0533] mRNA was isolated from a human tissue of interest using reagentsand protocols from Invitrogen, San Diego, Calif. (Fast Track 2). ThisRNA was used to generate an oligo dT primed cDNA library in the vectorpRK5D using reagents and protocols from Life Technologies, Gaithersburg,Md. (Super Script Plasmid System). In this procedure, the doublestranded cDNA was sized to greater than 1000 bp and the SalI/NotITinkered cDNA was cloned into XhoI/NotI cleaved vector. pRK5D is acloning vector that has an sp6 transcription initiation site followed byan SfiI restriction enzyme site preceding the XhoI/NotI cDNA cloningsites.

[0534] 2. Preparation of random primed cDNA library

[0535] A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

[0536] 3. Transformation and Detection

[0537] DNA from the library described in paragraph 2 above was chilledon ice to which was added electrocompetent DH10B bacteria (LifeTechnologies, 20 ml). The bacteria and vector mixture was thenelectroporated as recommended by the manufacturer. Subsequently, SOCmedia (Life Technologies, 1 ml) was added and the mixture was incubatedat 37° C. for 30 minutes. The transformants were then plated onto 20standard 150 mm LB plates containing ampicillin and incubated for 16hours (37° C.). Positive colonies were scraped off the plates and theDNA was isolated from the bacterial pellet using standard protocols,e.g. CsCl-gradient. The purified DNA was then carried on to the yeastprotocols below.

[0538] The yeast methods were divided into three categories: (1)Transformation of yeast with the plasmid/cDNA combined vector; (2)Detection and isolation of yeast clones secreting amylase; and (3) PCRamplification of the insert directly from the yeast colony andpurification of the DNA for sequencing and further analysis.

[0539] The yeast strain used was HD56-5A (ATCC-90785). This strain hasthe following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

[0540] Transformation was performed based on the protocol outlined byGietz et al., Nucl. Acid. Res., 20:1425 (1992). Transformed cells werethen inoculated from agar into YEPD complex media broth (100 ml) andgrown overnight at 30° C. The YEPD broth was prepared as described inKaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., p. 207 (1994). The overnight culture was thendiluted to about 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPDbroth (500 ml) and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4-0.5).

[0541] The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

[0542] Transformation took place by mixing the prepared cells (100 μl)with freshly denatured single stranded salmon testes DNA (LofstrandLabs, Gaithersburg, Md.) and transforming DNA (1 μg, vol.<10 μl) inmicrofuge tubes. The mixture was mixed briefly by vortexing, then 40%PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA,100 mM Li₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

[0543] Alternatively, instead of multiple small reactions, thetransformation was performed using a single, large scale reaction,wherein reagent amounts were scaled up accordingly.

[0544] The selective media used was a synthetic complete dextrose agarlacking uracil (SCD-Ura) prepared as described in Kaiser et al., Methodsin Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,p. 208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

[0545] The detection of colonies secreting amylase was performed byincluding red starch in the selective growth media. Starch was coupledto the red dye (Reactive Red-120, Sigma) as per the procedure describedby Biely et al., Anal. Biochem., 172:176-179 (1988). The coupled starchwas incorporated into the SCD-Ura agar plates at a final concentrationof 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

[0546] The positive colonies were picked and streaked across freshselective media (onto 150 mm plates) in order to obtain well isolatedand identifiable single colonies. Well isolated single colonies positivefor amylase secretion were detected by direct incorporation of redstarch into buffered SCD-Ura agar. Positive colonies were determined bytheir ability to break down starch resulting in a clear halo around thepositive colony visualized directly.

[0547] 4. Isolation of DNA by PCR Amplification

[0548] When a positive colony was isolated, a portion of it was pickedby a toothpick and diluted into sterile water (30 μl) in a 96 wellplate. At this time, the positive colonies were either frozen and storedfor subsequent analysis or immediately amplified. An aliquot of cells (5μl) was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Klentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water.

[0549] The sequence of the forward oligonucleotide 1 was:

[0550] 5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′ (SEQ ID NO:25)

[0551] The sequence of reverse oligonucleotide 2 was:

[0552] 5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′ (SEQ ID NO:26)

[0553] PCR was then performed as follows: a. Denature 92° C., 5 minutesb. 3 cycles of: Denature 92° C., 30 seconds Anneal 59° C., 30 secondsExtend 72° C., 60 seconds c. 3 cycles of: Denature 92° C., 30 secondsAnneal 57° C., 30 seconds Extend 72° C., 60 seconds d. 25 cycles of:Denature 92° C., 30 seconds Anneal 55° C., 30 seconds Extend 72° C., 60seconds e. Hold  4° C.

[0554] The underlined regions of the oligonucleotides annealed to theADH promoter region and the amylase region, respectively, and amplifieda 307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

[0555] Following the PCR, an aliquot of the reaction (5 μl) was examinedby agarose gel electrophoresis in a 1% agarose gel using aTris-Borate-EDTA (TBE) buffering system as described by Sambrook et al.,supra. Clones resulting in a single strong PCR product larger than 400bp were further analyzed by DNA sequencing after purification with a 96Qiaquick PCR clean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3

[0556] Isolation of cDNA Clones Using Signal Algorithm Analysis

[0557] Various polypeptide-encoding nucleic acid sequences wereidentified by applying a proprietary signal sequence finding algorithmdeveloped by Genentech, Inc. (South San Francisco, Calif.) upon ESTs aswell as clustered and assembled EST fragments from public (e.g.,GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals, Inc., PaloAlto, Calif.) databases. The signal sequence algorithm computes asecretion signal score based on the character of the DNA nucleotidessurrounding the first and optionally the second methionine codon(s)(ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

Example 4

[0558] Isolation of cDNA Clones Encoding Human PRO1800

[0559] A consensus DNA sequence was assembled relative to other ESTsequences using phrap as described in Example 1 above. This consensussequence is herein designated DNA30934. Based on the DNA30934 consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence forPRO1800.

[0560] PCR primers (forward and reverse) were synthesized: forward PCRprimer (30934.f1) 5′-GCATAATGGATGTCACTGAGG-3′ (SEQ ID NO:3) reverse PCRprimer (30934.r1) 5′-AGAACAATCCTGCTGAAAGCTAG-3′ (SEQ ID NO:4)

[0561] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30934 sequence which had the followingnucleotide sequence hybridization probe (30934.p1)5′-GAAACGAGGAGGCGGCTCAGTGGTGATCGTGTCTTCCATAGCAGCC-3′ (SEQ ID NO:5)

[0562] RNA for construction of the cDNA libraries was isolated fromhuman fetal liver tissue. DNA sequencing of the clones isolated asdescribed above gave the full-length DNA sequence for PRO1800(designated herein as DNA35672-2508 [FIG. 1, SEQ ID NO:1]; and thederived protein sequence for PRO1800.

[0563] The entire nucleotide sequence of DNA35672-2508 is shown in FIG.1 (SEQ ID NO:1). Clone DNA35672-2508 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 36-38 and ending at the stop codon at nucleotide positions870-872 (FIG. 1). The predicted polypeptide precursor is 278 amino acidslong (FIG. 2). The full-length PRO1800 protein shown in FIG. 2 has anestimated molecular weight of about 29,537 daltons and a pl of about8.97. Analysis of the full-length PRO1800 sequence shown in FIG. 2 (SEQID NO:2) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 15, a potential N-glycosylationsite from about amino acid 183 to about amino acid 186, potentialN-myristolation sites from about amino acid 43 to about amino acid 48,from about amino acid 80 to about amino acid 85, from about amino acid191 to about amino acid 196, from about amino acid 213 to about aminoacid 218 and from about amino acid 272 to about amino acid 277 and amicrobodies C-terminal targeting signal from about amino acid 276 toabout amino acid 278. Clone DNA35672-2508 has been deposited with ATCCon Dec. 15, 1998 and is assigned ATCC deposit no. 203538.

[0564] An analysis of the Dayhoff database (version 35.45 SwissProt 35),using a WU-BLAST2 sequence alignment analysis of the full-lengthsequence shown in FIG. 2 (SEQ ID NO:2), evidenced significant homologybetween the PRO1800 amino acid sequence and the following Dayhoffsequences: HE27_HUMAN, CELF36H9_(—)1, CEF54F3_(—)3, A69621,AP000007_(—)227, UCPA_ECOLI, F69868, Y4LA_RHISN DHK2_STRVN andDHG1_BACME.

Example 5

[0565] Isolation of cDNA Clones Encoding Human PRO539

[0566] A consensus DNA sequence was assembled relative to other ESTsequences using phrap as described in Example 1. This consensus sequenceis herein designated DNA41882. Based on the DNA41882 consensus sequenceshown, oligonucleotides were synthesized: 1) to identify by PCR a cDNAlibrary that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO539.

[0567] RNA for construction of the cDNA libraries was isolated fromhuman fetal kidney tissue. DNA sequencing of the clones isolated asdescribed above gave the full-length DNA sequence for PRO539 (designatedherein as DNA47465-1561 [FIG. 3, SEQ ID NO:6]; and the derived proteinsequence for PRO539.

[0568] The entire nucleotide sequence of DNA47465-1561 is shown in FIG.3 (SEQ ID NO:6). Clone DNA47465-1561 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 186-188 and ending at the stop codon at nucleotide positions2676-2678 (FIG. 3). The predicted polypeptide precursor is 830 aminoacids long (FIG. 4). The full-length PRO539 protein shown in FIG. 4 hasan estimated molecular weight of about 95,029 daltons and a pl of about8.26. Analysis of the full-length PRO539 sequence shown in FIG. 4 (SEQID NO:7) evidences the presence of the following: leucine zipper patternsequences from about amino acid 557 to about amino acid 578 and fromabout amino acid 794 to about amino acid 815, potential N-glycosylationsites from about amino acid 133 to about amino acid 136 and from aboutamino acid 383 to about amino acid 386 and a kinesin-related proteinKif-4 coiled coil domain from about amino acid 231 to about amino acid672. Clone DNA47465-1561 has been deposited with ATCC on Feb. 9, 1999and is assigned ATCC deposit no. 203661.

[0569] An analysis of the Dayhoff database (version 35.45 SwissProt 35),using a WU-BLAST2 sequence alignment analysis of the full-lengthsequence shown in FIG. 4 (SEQ ID NO:7), evidenced homology between thePRO539 amino acid sequence and the following Dayhoff sequences:AF019250_(—)1, KIF4_MOUSE, TRHY_HUMAN, A56514, G02520, MYSP_HUMAN,AF041382_(—)1, A45592, HS125H2_(—)1 and HS6802_(—)2.

Example 6

[0570] Isolation of cDNA Clones Encoding Human PRO982

[0571] Use of the signal sequence algorithm described in Example 3 aboveallowed identification of a single Incyle EST sequence designated hereinas Incyte EST cluster sequence no. 43715. This EST sequence was comparedto a variety of EST databases which included public EST databases (e.g.,GenBank) and a proprietary EST DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) to identify existing homologies. Thehomology search was performed using the computer program BLAST or BLAST2(Altshul et al., Methods in Enzymology 266:460-480 (1996)). Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto a consensus DNA sequence with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.). The consensus sequenceobtained therefrom is designated DNA56095.

[0572] In light of an observed sequence homology between DNA56095 andMerck EST no. AA024389, Merck EST clone AA024389 was obtained andsequenced. The sequence, designated DNA57700-1408 (SEQ ID NO:8), isshown in FIG. 5. It is the full-length DNA sequence for PRO982.

[0573] The full length clone shown in FIG. 5 contains a single openreading frame with an apparent translational initiation site atnucleotide positions 26-28 and ending at the stop codon found atnucleotide positions 401-403 (SEQ ID NO:8). The predicted polypeptideprecursor is 125 amino acids long, has a calculated molecular weight ofapproximately 14,198 daltons and an estimated pI of approximately 9.01.Analysis of the full-length PRO982 sequence shown in FIG. 6 (SEQ IDNO:9) evidences the presence of a signal peptide from about amino acid 1to about amino acid 21 and potential anaphylatoxin domain from aboutamino acid 50 to about amino acid 59. An analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced homology between thePRO982 amino acid sequence and the following Dayhoff sequences:RNTMDCV_(—)1; A48151; WAP_RAT; S24596;A53640;MT4_HUMAN; U93486_(—)1;SYNBILGFG_(—)1; P_R49917; and P_R41880. Clone DNA57700-1408 wasdeposited with the ATCC on Jan. 12, 1999 and is assigned ATCC depositno. 203583.

Example 7

[0574] Isolation of cDNA Clones Encoding Human PRO1434

[0575] A consensus DNA sequence was assembled relative to other ESTsequences using phrap as described in Example 1 above. This consensussequence is herein designated DNA54187. Based on the DNA54187 consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence forPRO1434.

[0576] PCR primers (forward and reverse) were synthesized: forward PCRprimer 5′-GAGGTGTCGCTGTGAAGCCAACGG-3′ (SEQ ID NO:12) reverse PCR primer5′-CGCTCGATTCTCCATGTGCCTTCC-3′ (SEQ ID NO:13)

[0577] Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA54187 sequence which had the followingnucleotide sequence hybridization probe5′-GACGGAGTGTGTGGACCCTGTGTACGAGCCTGATCAGTGCTGTCC-3′ (SEQ ID NO:14)

[0578] RNA for construction of the cDNA libraries was isolated fromhuman retina tissue (LIB94). DNA sequencing of the clones isolated asdescribed above gave the full-length DNA sequence for PRO1434(designated herein as DNA68818-2536 [FIG. 7, SEQ ID NO:10]; and thederived protein sequence for PRO1434.

[0579] The entire nucleotide sequence of DNA68818-2536 is shown in FIG.7 (SEQ ID NO:10). Clone DNA68818-2536 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 581-583 and ending at the stop codon at nucleotide positions1556-1558 (FIG. 7). The predicted polypeptide precursor is 325 aminoacids long (FIG. 8). The full-length PRO1434 protein shown in FIG. 8 hasan estimated molecular weight of about 35,296 daltons and a pl of about5.37. Analysis of the full-length PRO1434 sequence shown in FIG. 8 (SEQID NO:11) evidences the presence of a variety of important proteindomains as shown in FIG. 8. Clone DNA68818-2536 has been deposited withATCC on Feb. 9, 1999 and is assigned ATCC deposit no. 203657.

[0580] An analysis of the Dayhoff database (version 35.45 SwissProt 35),using a WU-BLAST2 sequence alignment analysis of the full-lengthsequence shown in FIG. 8 (SEQ ID NO:11), evidenced significant homologybetween the PRO1434 amino acid sequence and the following Dayhoffsequences: NEL_MOUSE,APMU_PIG,P_W37501,NEL_RAT,TSP1_CHICK,P_W37500,NEL2_HUMAN,MMU010792_(—)1,D86983_(—)1and 10 MUCS_BOVIN.

Example 8

[0581] Isolation of cDNA Clones Encoding Human PRO1863

[0582] Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase, designated Incyte EST cluster sequence no. 82468. This ESTcluster sequence was then compared to a variety of expressed sequencetag (EST) databases which included public EST databases (e.g., GenBank)and a proprietary EST DNA database (Lifeseq®, Incyte Pharmaceuticals,Palo Alto, Calif.) to identify existing homologies. The homology searchwas performed using the computer program BLAST or BLAST2 (Altshul etal., Methods in Enzymology 266:460-480 (1996)). Those comparisonsresulting in a BLAST score of 70 (or in some cases 90) or greater thatdid not encode known proteins were clustered and assembled into aconsensus DNA sequence with the program “phrap” (Phil Green, Universityof Washington, Seattle, Wash.). The consensus sequence obtainedtherefrom is herein designated DNA56029.

[0583] In light of the sequence homology between the DNA56029 sequenceand an EST sequence contained within the Incyte EST clone no. 2186536,the Incyte EST clone no. 2186536 was purchased and the cDNA insert wasobtained and sequenced. The sequence of this cDNA insert is shown inFIG. 9 and is herein designated as DNA59847-2510.

[0584] Clone DNA59847-2510 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 17-19 andending at the stop codon at nucleotide positions 1328-1330 (FIG. 9). Thepredicted polypeptide precursor is 437 amino acids long (FIG. 10). Thefull-length PRO1863 protein shown in FIG. 10 has an estimated molecularweight of about 46,363 daltons and a pI of about 6.22. Analysis of thefull-length PRO1863 sequence shown in FIG. 10 (SEQ ID NO:16) evidencesthe presence of the following: a signal peptide from about amino acid 1to about amino acid 15, a transmembrane domain from about amino acid 243to about amino acid 260, potential N-glycosylation sites from aboutamino acid 46 to about amino acid 49, from about amino acid 189 to aboutamino acid 192 and from about amino acid 382 to about amino acid 385,glycosaminoglycan attachment sites from about amino acid 51 to aboutamino acid 54 and from about amino acid 359 to about amino acid 362 andpotential N-myristolation sites from about amino acid 54 to about aminoacid 59, from about amino acid 75 to about amino acid 80, from aboutamino acid 141 to about amino acid 146, from about amino acid 154 toabout amino acid 159, from about amino acid 168 to about amino acid 173,from about amino acid 169 to about amino acid 174, from about amino acid198 to about amino acid 203, from about amino acid 254 to about aminoacid 259, from about amino acid 261 to about amino acid 266, from aboutamino acid 269 to about amino acid 274, from about amino acid 284 toabout amino acid 289, from about amino acid 333 to about amino acid 338,from about amino acid 347 to about amino acid 352, from about amino acid360 to about amino acid 365, from about amino acid 361 to about aminoacid 366, from about amino acid 388 to about amino acid 393, from aboutamino acid 408 to about amino acid 413 and from about amino acid 419 toabout amino acid 424. Clone DNA59847-2510 has been deposited with ATCCon Jan. 12, 1999 and is assigned ATCC deposit no. 203576.

[0585] An analysis of the Dayhoff database (version 35.45 SwissProt 35),using a WU-BLAST2 sequence alignment analysis of the full-lengthsequence shown in FIG. 10 (SEQ ID NO:16), evidenced homology between thePRO1863 amino acid sequence and the following Dayhoff sequences:AF041083_(—)1, P_W26579, HSA223603_(—)1, MMU97068, RNMAGPIAN_(—)1,CAHX_FLABR, S61882, AB007899_(—)1, CAH1_FLALI and P_W13386.

Example 9

[0586] Isolation of cDNA Clones Encoding Human PRO1917

[0587] Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the LIFESEQ®database, designated EST cluster no. 85496. This EST cluster sequencewas then compared to a the EST databases listed above to identifyexisting homologies. The homology search was performed using thecomputer program BLAST or BLAST2 (Altshul et al., Methods in Enzymology266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70(or in some cases 90) or greater that did not encode known proteins wereclustered and assembled into a consensus DNA sequence with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.). Theconsensus sequence obtained therefrom is herein designated DNA56415.

[0588] In light of the sequence homology between the DNA56415 sequenceand an EST sequence contained within EST no.3255033, the EST clone,which derived from an ovarian tumor library, was purchased and the cDNAinsert was obtained and sequenced. The sequence of this cDNA insert isshown in FIG. 11 and is herein designated as DNA76400-2528.

[0589] The full length clone shown in FIG. 11 contained a single openreading frame with an apparent translational initiation site atnucleotide positions 6-9 and ending at the stop codon found atnucleotide positions 1467-1469 (FIG. 11; SEQ ID NO:17). The predictedpolypeptide precursor (FIG. 12, SEQ ID NO:18) is 487 amino acids long.PRO1917 has a calculated molecular weight of approximately 55,051daltons and an estimated pI of approximately 8.14. Additional featuresinclude: a signal peptide at about amino acids 1-30; potentialN-glycosylation sites at about amino acids 242-245 and 481-484, proteinkinase C phosphorylation sites at about amino acids 95-97, 182-184, and427-429; N-myristoylation sites at about amino acids 107-112, 113-118,117-122, 118-123, and 128-133; and an endoplasmic reticulum targetingsequence at about amino acids 484-487.

[0590] An analysis of the Dayhoff database (version 35.45 SwissProt 35),using a WU-BLAST2 sequence alignment analysis of the full-lengthsequence shown in FIG. 12 (SEQ ID NO:18), revealed significant homologybetween the PRO1917 amino acid sequence and Dayhoff sequenceAF012714_(—)1. Significant homology was also revealed between thePRO1917 amino acid sequence and the sequence of a chondrocyte protein,designated “P_W52286” on the Dayhoff database, which has been reportedto be involved in the transition of chondrocytes from proliferate tohypertrophic states (International Patent Application Publication No.WO9801468-A1). Homology was also revealed between the PRO1917 amino acidsequence and the following additional Dayhoff sequences: P_W52286,GGU59420_(—)1, P_R25597, PPA3_YEAST, PPA1_SCHPO, PPA2_SCHPO,A46783_(—)1, DMC165H7_(—)1, and AST8_DROME.

[0591] Clone DNA76400-2528 was deposited with the ATCC on Jan. 12, 1999,and is assigned ATCC deposit no. 203573.

Example 10

[0592] Isolation of cDNA Clones Encoding Human PRO1868

[0593] A consensus DNA sequence was assembled relative to other ESTsequences using phrap as described in Example 1 above. This consensussequence is herein designated DNA49803. Based up an observed homologybetween the DNA49803 consensus sequence and an EST sequence containedwithin the Incyte EST clone no. 2994689, Incyte EST clone no. 2994689was purchased and its insert obtained and sequenced. The sequence ofthat insert is shown in FIG. 13 and is herein designated DNA77624-2515.

[0594] The entire nucleotide sequence of DNA77624-2515 is shown in FIG.13 (SEQ ID NO:19). Clone DNA77624-2515 contains a single open readingframe with an apparent translational initiation site at nucleotidepositions 51-53 and ending at the stop codon at nucleotide positions981-983 (FIG. 13). The predicted polypeptide precursor is 310 aminoacids long (FIG. 14). The full-length PRO1868 protein shown in FIG. 14has an estimated molecular weight of about 35,020 daltons and a pI ofabout 7.90. Analysis of the full-length PRO1868 sequence shown in FIG.14 (SEQ ID NO:20) evidences the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 30, a transmembranedomain from about amino acid 243 to about amino acid 263, potentialN-glycosylation sites from about amino acid 104 to about amino acid 107and from about amino acid 192 to about amino acid 195, a cAMP- andcGMP-dependent protein kinase phosphorylation site from about amino acid107 to about amino acid 110, casein kinase II phosphorylation sites fromabout amino acid 106 to about amino acid 109 and from about amino acid296 to about amino acid 299, a tyrosine kinase phosphorylation site fromabout amino acid 69 to about amino acid 77 and potential N-myristolationsites from about amino acid 26 to about amino acid 31, from about aminoacid 215 to about amino acid 220, from about amino acid 226 to aboutamino acid 231, from about amino acid 243 to about amino acid 248, fromabout amino acid 244 to about amino acid 249 and from about amino acid262 to about amino acid 267. Clone DNA77624-2515 has been deposited withATCC on Dec. 22, 1998 and is assigned ATCC deposit no. 203553.

[0595] An analysis of the Dayhoff database (version 35.45 SwissProt 35),using a WU-BLAST2 sequence alignment analysis of the full-lengthsequence shown in FIG. 14 (SEQ ID NO:20), evidenced significant homologybetween the PRO1868 amino acid sequence and the following Dayhoffsequences: HGS_RC75, P_W61379, A33_HUMAN, P_W14146, P_W14158,AMAL_DROME, P_R77437, I38346, NCM2_HUMAN and PTPD_HUMAN.

Example 11

[0596] Isolation of cDNA Clones Encoding Human PRO3434

[0597] Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (Lifeseq®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA56009.

[0598] In light of the sequence homology between the DNA56009 sequenceand an EST sequence contained within the Incyte EST clone no. 3327089,the Incyte EST clone no. 3327089 was purchased and the cDNA insert wasobtained and sequenced. The sequence of this cDNA insert is shown inFIG. 15 and is herein designated as DNA77631-2537.

[0599] Clone DNA77631-2537 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 46-48 andending at the stop codon at nucleotide positions 3133-3135 (FIG. 15).The predicted polypeptide precursor is 1029 amino acids long (FIG. 16).The full-length PRO3434 protein shown in FIG. 16 has an estimatedmolecular weight of about 114,213 daltons and a pI of about 6.42.Analysis of the full-length PRO3434 sequence shown in FIG. 16 (SEQ IDNO:22) evidences the presence of very important polypeptide domains asshown in FIG. 16. Clone DNA77631-2537 has been deposited with ATCC onFeb. 9, 1999 and is assigned ATCC deposit no. 203651.

[0600] An analysis of the Dayhoff database (version 35.45 SwissProt 35),using a WU-BLAST2 sequence alignment analysis of the full-lengthsequence shown in FIG. 16 (SEQ ID NO:22), evidenced homology between thePRO3434 amino acid sequence and the following Dayhoff sequences:VATX_YEAST, P_R51171, POLS_IBDVP, IBDVORF_(—)2. JC5043, IBDVPIV_(—)1,VE7_HPV11, GEN14220, MUTS_THETH and COAC_CHICK.

Example 12

[0601] Isolation of cDNA Clones Encoding Human PRO1927

[0602] Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the LIFESEQ®database, designated EST Cluster No. 1913. This EST cluster sequence wasthen compared to a variety of expressed sequence tag (EST) databaseswhich included the databases listed above, including an additionalproprietary EST DNA database (Genentech, South San Francisco, Calif.) toidentify existing homologies. The homology search was performed usingthe computer program BLAST or BLAST2 (Altshul et al., Methods inEnzymology 266:460-480 (1996)). Those comparisons resulting in a BLASTscore of 70 (or in some cases 90) or greater that did not encode knownproteins were clustered and assembled into a consensus DNA sequence withthe program “phrap” (Phil Green, University of Washington, Seattle,Wash.). The consensus sequence obtained therefrom is herein designatedDNA73896.

[0603] In light of the sequence homology between the DNA73896 sequenceand an EST sequence contained within EST no.3326981H1, EST clone no.3326981H1, which was obtained from a library constructed from RNAisolated from aortic tissue, was purchased and the cDNA insert wasobtained and sequenced. The sequence of this cDNA insert is shown inFIG. 17 and is herein designated as “DNA82307-2531”.

[0604] The full length clone shown in FIG. 17 contained a single openreading frame with an apparent translational initiation site atnucleotide positions 51-53 and ending at the stop codon found atnucleotide positions 1695-1697 (FIG. 17; SEQ ID NO:23). The predictedpolypeptide precursor (FIG. 18, SEQ ID NO:24) is 548 amino acids long.PRO1927 has a calculated molecular weight of approximately 63,198daltons and an estimated pI of approximately 8.10. Additional featuresinclude: a signal peptide at about amino acids 1-23; a putativetransmembrane domain at about amino acids 6-25; potentialN-glycosylation sites at about amino acids 5-8, 87-90, 103-106, and465-469; potential N-myristoylation sites at about amino acids 6-11,136-141, 370-375, and 509-514.

[0605] An analysis of the Dayhoff database (version 35.45 SwissProt 35),using a WU-BLAST2 sequence alignment analysis of the full-lengthsequence shown in FIG. 18 (SEQ ID NO:24), revealed significant homologybetween the PRO1927 amino acid sequence and Dayhoff sequenceAB000628_(—)1. Homology was also revealed between the PRO1927 amino acidsequence and the following additional Dayhoff sequences: HGS_A251,HGS_A197, CELC50H11_(—)2, CPXM_BACSU, VF03_VACCC, VF03_VACCV,DYHA_CHLRE,C69084, and A64315.

[0606] Clone DNA82307-2531 was deposited with the ATCC on Dec. 15, 1998,and is assigned ATCC deposit no. 203537.

Example 13

[0607] Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay(Assay 67)

[0608] This example shows that one or more of the polypeptides of theinvention are active as inhibitors of the proliferation of stimulatedT-lymphocytes. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of an immune response isbeneficial.

[0609] The basic protocol for this assay is described in CurrentProtocols in Immunology, unit 3.12; edited by J E Coligan, A MKruisbeek, D H Marglies, E M Shevach, W Strober, National Insitutes ofHealth, Published by John Wiley & Sons, Inc.

[0610] More specifically, in one assay variant, peripheral bloodmononuclear cells (PBMC) are isolated from mammalian individuals, forexample a human volunteer, by leukopheresis (one donor will supplystimulator PBMCs, the other donor will supply responder PBMCs). Ifdesired, the cells are frozen in fetal bovine serum and DMSO afterisolation. Frozen cells may be thawed overnight in assay media (37° C.,5% CO₂) and then washed and resuspended to 3×10⁶ cells/ml of assay media(RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine,1% HEPES, 1% non-essential amino acids, 1 % pyruvate). The stimulatorPBMCs are prepared by irradiating the cells (about 3000 Rads).

[0611] The assay is prepared by plating in triplicate wells a mixtureof:

[0612] 100:1 of test sample diluted to 1% or to 0.1%,

[0613] 50:1 of irradiated stimulator cells, and

[0614] 50:1 of responder PBMC cells.

[0615] 100 microliters of cell culture media or 100 microliter ofCD4-IgG is used as the control. The wells are then incubated at 37° C.,5% CO₂ for 4 days. On day 5, each well is pulsed with tritiatedthymidine (1.0 mC/well; Amersham). After 6 hours the cells are washed 3times and then the uptake of the label is evaluated.

[0616] In another variant of this assay, PBMCs are isolated from thespleens of Balb/c mice and C57B6 mice. The cells are teased from freshlyharvested spleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

[0617] Any decreases below control is considered to be a positive resultfor an inhibitory compound, with decreases of less than or equal to 80%being preferred. However, any value less than control indicates aninhibitory effect for the test protein.

[0618] The following polypeptides tested positive in this assay: PRO1917and PRO1868.

Example 14

[0619] Skin Vascular Permeability Assay (Assay 64)

[0620] This assay shows that certain polypeptides of the inventionstimulate an immune response and induce inflammation by inducingmononuclear cell, eosinophil and PMN infiltration at the site ofinjection of the animal. Compounds which stimulate an immune responseare useful therapeutically where stimulation of an immune response isbeneficial. This skin vascular permeability assay is conducted asfollows. Hairless guinea pigs weighing 350 grams or more areanesthetized with ketamine (75-80 mg/Kg) and 5 mg/Kg xylazineintramuscularly (IM). A sample of purified polypeptide of the inventionor a conditioned media test sample is injected intradermally onto thebacks of the test animals with 100 μl per injection site. It is possibleto have about 10-30, preferably about 16-24, injection sites per animal.One μl of Evans blue dye (1% in physiologic buffered saline) is injectedintracardially. Blemishes at the injection sites are then measured (mmdiameter) at 1 hr and 6 hr post injection. Animals were sacrificed at 6hrs after injection. Each skin injection site is biopsied and fixed informalin. The skins are then prepared for histopathologic evaluation.Each site is evaluated for inflammatory cell infiltration into the skin.Sites with visible inflammatory cell inflammation are scored aspositive. Inflammatory cells may be neutrophilic, eosinophilic,monocytic or lymphocytic. At least a minimal perivascular infiltrate atthe injection site is scored as positve, no infiltrate at the site ofinjection is scored as negative.

[0621] The following polypeptides tested positive in this assay:PRO1434.

Example 15

[0622] Proliferation of Rat Utricular Supporting Cells (Assay 54)

[0623] This assay shows that certain polypeptides of the invention actas potent mitogens for inner ear supporting cells which are auditoryhair cell progenitors and, therefore, are useful for inducing theregeneration of auditory hair cells and treating hearing loss inmammals. The assay is performed as follows. Rat UEC-4 utricularepithelial cells are aliquoted into 96 well plates with a density of3000 cells/well in 200 μl of serum-containing medium at 33° C. The cellsare cultured overnight and are then switched to serum-free medium at 37°C. Various dilutions of PRO polypeptides (or nothing for a control) arethen added to the cultures and the cells are incubated for 24 hours.After the 24 hour incubation, ³H-thymidine (1 μCi/well) is added and thecells are then cultured for an additional 24 hours. The cultures arethen washed to remove unincorporated radiolabel, the cells harvested andCpm per well determined. Cpm of at least 30% or greater in the PROpolypeptide treated cultures as compared to the control cultures isconsidered a positive in the assay.

[0624] The following polypeptides tested positive in this assay: PRO982.

Example 16

[0625] Gene Amplification

[0626] This example shows that the PRO1800-, PRO539-, PRO3434- andPRO1927-encoding genes are amplified in the genome of certain humanlung, colon and/or breast cancers and/or cell lines. Amplification isassociated with overexpression of the gene product, indicating that thepolypeptides are useful targets for therapeutic intervention in certaincancers such as colon, lung, breast and other cancers and diagnosticdetermination of the presence of those cancers. Therapeutic agents maytake the form of antagonists of PRO1800, PRO539, PRO3434 or PRO1927polypeptide, for example, murine-human chimeric, humanized or humanantibodies against a PRO1800, PRO539, PRO3434 or PRO1927 polypeptide.

[0627] The starting material for the screen was genomic DNA isolatedfrom a variety cancers. The DNA is quantitated precisely, e.g.,fluorometrically. As a negative control, DNA was isolated from the cellsof ten normal healthy individuals which was pooled and used as assaycontrols for the gene copy in healthy individuals (not shown). The 5′nuclease assay (for example, TaqMan™) and real-time quantitative PCR(for example, ABI Prizm 7700 Sequence Detection System™ (Perkin Elmer,Applied Biosystems Division, Foster City, Calif.)), were used to findgenes potentially amplified in certain cancers. The results were used todetermine whether the DNA encoding PRO1800, PRO539, PRO3434 or PRO1927is over-represented in any of the primary lung or colon cancers orcancer cell lines or breast cancer cell lines that were screened. Theprimary lung cancers were obtained from individuals with tumors of thetype and stage as indicated in Table 6. An explanation of theabbreviations used for the designation of the primary tumors listed inTable 6 and the primary tumors and cell lines referred to throughoutthis example are given below.

[0628] The results of the TaqMan™ are reported in delta (Δ) Ct units.One unit corresponds to 1 PCR cycle or approximately a 2-foldamplification relative to normal, two units corresponds to 4-fold, 3units to 8-fold amplification and so on. Quantitation was obtained usingprimers and a TaqMan™ fluorescent probe derived from the PRO1800-,PRO539-, PRO3434- or PRO1927-encoding gene. Regions of PRO1800, PRO539,PRO3434 or PRO1927 which are most likely to contain unique nucleic acidsequences and which are least likely to have spliced out introns arepreferred for the primer and probe derivation, e.g., 3′-untranslatedregions. The sequences for the primers and probes (forward, reverse andprobe) used for the PRO1800, PRO539, PRO3434 or PRO1927 geneamplification analysis were as follows:

[0629] PRO1800 (DNA35672-2508) forward 5′-ACTCGGGATTCCTGCTGTT-3′ (SEQ IDNO:27) probe 5′-AGGCCTTTACCCAAGGCCACAAC-3′ (SEQ ID NO:28) reverse5′-GGCCTGTCCTGTGTTCTCA-3′ (SEQ ID NO:29)

[0630] PRO539 (DNA47465-1561) forward 5′-TCCCACCACTTACTTCCATGAA-3′ (SEQID NO:30) probe 5′-CTGTGGTACCCAATTGCCGCCTTGT-3′ (SEQ ID NO:31) reverse5′-ATTGTCCTGAGATTCGAGCAAGA-3′ (SEQ ID NO:32)

[0631] PRO3434 (DNA77631-2537) forward 5′-GTCCAGCAAGCCCTCATT-3′ (SEQ IDNO:33) probe 5′-CTTCTGGGCCACAGCCCTGC-3′ (SEQ ID NO:34) reverse5′-CAGTTCAGGTCGTTTCACA-3′ (SEQ ID NO:35)

[0632] PRO1927 (DNA82307-2531) forward 5′-CCAGTCAGGCCGTTTTAGA-3′ (SEQ IDNO:36) probe 5′-CGGGCGCCCAAGTAAAAGCTC-3′ (SEQ ID NO:37) reverse5′-CATAAAGTAGTATATGCATTCCAGTGTT-3′ (SEQ ID NO:38)

[0633] The 5′ nuclease assay reaction is a fluorescent PCR-basedtechnique which makes use of the 5′ exonuclease activity of Taq DNApolymerase enzyme to monitor amplification in real time. Twooligonucleotide primers are used to generate an amplicon typical of aPCR reaction. A third oligonucleotide, or probe, is designed to detectnucleotide sequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

[0634] The 5′ nuclease procedure is run on a real-time quantitative PCRdevice such as the ABI Prism 7700TM Sequence Detection. The systemconsists of a thermocycler, laser, charge-coupled device (CCD) cameraand computer. The system amplifies samples in a 96-well format on athermocycler. During amplification, laser-induced fluorescent signal iscollected in real-time through fiber optics cables for all 96 wells, anddetected at the CCD. The system includes software for running theinstrument and for analyzing the data.

[0635] 5′ Nuclease assay data are initially expressed as Ct, or thethreshold cycle. This is defined as the cycle at which the reportersignal accumulates above the background level of fluorescence. The ΔCtvalues are used as quantitative measurement of the relative number ofstarting copies of a particular target sequence in a nucleic acid samplewhen comparing cancer DNA results to normal human DNA results.

[0636] Table 6 describes the stage, T stage and N stage of variousprimary tumors which were used to screen the PRO1800, PRO539, PRO3434and PRO1927 compounds of the invention. TABLE 6 Primary Lung and ColonTumor Profiles Other Dukes T N Primary Tumor Stage Stage Stage StageStage Stage Human lung tumor AdenoCa IIA T1 N1 (SRCC724) [LT1] Humanlung tumor SqCCa IIB T3 N0 (SRCC725) [LT1a] Human lung tumor AdenoCa IBT2 N0 (SRCC726) [LT2] Human lung tumor AdenoCa IIIA T1 N2 (SRCC727)[LT3] Human lung tumor AdenoCa IB T2 N0 (SRCC728) [LT4] Human lung tumorSqCCa IB T2 N0 (SRCC729) [LT6] Human lung tumor Aden/SqCCa IA T1 N0(SRCC730) [LT7] Human lung tumor AdenoCa IB T2 N0 (SRCC731) [LT9] Humanlung tumor SqCCa IIB T2 N1 (SRCC732) [LT10] Human lung tumor SqCCa IIAT1 N1 (SRCC733) [LT11] Human lung tumor AdenoCa IV T2 N0 (SRCC734)[LT12] Human lung tumor AdenoSqCCa IB T2 N0 (SRCC735) [LT13] Human lungtumor SqCCa IB T2 N0 (SRCC736) [LT15] Human lung tumor SqCCa IB T2 N0(SRCC737) [LT16] Human lung tumor SqCCa IIB T2 N1 (SRCC738) [LT17] Humanlung tumor SqCCa IB T2 N0 (SRCC739) [LT18] Human lung tumor SqCCa IB T2N0 (SRCC740) [LT19] Human lung tumor LCCa IIB T3 N1 (SRCC741) [LT21]Human lung AdenoCa IA T1 N0 (SRCC811) [LT22] Human colon AdenoCa M1 DpT4 N0 (SRCC742) [CT2] Human colon AdenoCa B pT3 N0 (SRCC743) [CT3]Human colon AdenoCa B T3 N0 (SRCC744) [CT8] Human colon AdenoCa A pT2 N0(SRCC745) [CT10] Human colon AdenoCa MO, B T3 N0 (SRCC746) [CT12] R1Human colon AdenoCa pMO, B pT3 pN0 (SRCC747) [CT14] RO Human colonAdenoCa M1, D T4 N2 (SRCC748) [CT15] R2 Human colon AdenoCa pMO B pT3pN0 (SRCC749) [CT16] Human colon AdenoCa C1 pT3 pN1 (SRCC750) [CT17]Human colon AdenoCa MO, B pT3 N0 (SRCC751) [CT1] R1 Human colon AdenoCaB pT3 N0 (SRCC752) [CT4] Human colon AdenoCa G2 C1 pT3 pN0 (SRCC753)[CT5] Human colon AdenoCa pMO, B pT3 pN0 (SRCC754) [CT6] RO Human colonAdenoCa G1 A pT2 pN0 (SRCC755) [CT7] Human colon AdenoCa G3 D pT4 pN2(SRCC756) [CT9] Human colon AdenoCa B T3 N0 (SRCC757) [CT11] Human colonAdenoCa MO, B pT3 pN0 (SRCC758) [CT18] RO

[0637] DNA Preparation

[0638] DNA was prepared from cultured cell lines, primary tumors, normalhuman blood. The isolation was performed using purification kit, bufferset and protease and all from Quiagen, according to the manufacturer'sinstructions and the description below.

[0639] Cell culture lysis:

[0640] Cells were washed and trypsinized at a concentration of 7.5×10⁸per tip and pelleted by centrifuging at 1000 rpm for 5 minutes at 4° C.,followed by washing again with ½ volume of PBS recentrifugation. Thepellets were washed a third time, the suspended cells collected andwashed 2× with PBS. The cells were then suspended into 10 ml PBS. BufferC1 was equilibrated at 4° C. Qiagen protease #19155 was diluted into6.25 ml cold ddH₂O to a final concentration of 20 mg/ml and equilibratedat 4° C. 10 ml of G2 Buffer was prepar by diluting Qiagen RNAse A stock(100 mg/ml) to a final concentration of 200 μg/ml.

[0641] Buffer C1 (10 ml, 4° C.) and ddH2O (40 ml, 4° C.) were then addedto the 10 ml of cell suspension, mixed by inverting and incubated on icefor 10 minutes. The cell nuclei were pelleted by centrifuging in aBeckman swinging bucket rotor at 2500 rpm at 4° C. for 15 minutes. Thesupernatant was discarded and the nuclei were suspended with a vortexinto 2 ml Buffer C1 (at 4° C.) and 6 ml ddH₂O, followed by a second 4°C. centrifugation at 2500 rpm for 15 minutes. The nuclei were thenresuspended into the residual buffer using 200 μl per tip. G2 buffer (10ml) was added to the suspended nuclei while gentle vortexing wasapplied. Upon completion of buffer addition, vigorous vortexing wasapplied for 30 seconds. Quiagen protease (200 μl, prepared as indicatedabove) was added and incubated at 50° C. for 60 minutes. The incubationand centrifugation was repeated until the lysates were clear (e.g.,incubating additional 30-60 minutes, pelleting at 3000×g for 10 min., 4°C.).

[0642] Solid human tumor sample preparation and lysis:

[0643] Tumor samples were weighed and placed into 50 ml conical tubesand held on ice. Processing was limited to no more than 250 mg tissueper preparation (1 tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 mg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock).The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood in order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at 2×30 seconds each in 2LddH₂O, followed by G2 buffer (50 ml). If tissue was still present on thegenerator tip, the apparatus was disassembled and cleaned.

[0644] Quiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10min., 4° C.).

[0645] Human blood preparation and lysis:

[0646] Blood was drawn from healthy volunteers using standard infectiousagent protocols and citrated into 10 ml samples per tip. Quiagenprotease was freshly prepared by dilution into 6.25 ml cold ddH₂O to afinal concentration of 20 mg/ml and stored at 4° C. G2 buffer wasprepared by diluting RNAse A to a final concentration of 200 μg/ml from100 mg/ml stock. The blood (10 ml) was placed into a 50 ml conical tubeand 10 ml C1 buffer and 30 ml ddH₂O (both previously equilibrated to 4°C.) were added, and the components mixed by inverting and held on icefor 10 minutes. The nuclei were pelleted with a Beckman swinging bucketrotor at 2500 rpm, 4° C. for 15 minutes and the supernatant discarded.With a vortex, the nuclei were suspended into 2 ml C1 buffer (4° C.) and6 ml ddH₂O (4° C.). Vortexing was repeated until the pellet was white.The nuclei were then suspended into the residual buffer using a 200 μltip. G2 buffer (10 ml) were added to the suspended nuclei while gentlyvortexing, followed by vigorous vortexing for 30 seconds. Quiagenprotease was added (200 μl) and incubated at 50° C. for 60 minutes. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10min., 4° C.).

[0647] Purification of cleared lysates:

[0648] (1) Isolation of genomic DNA:

[0649] Genomic DNA was equilibrated (1 sample per maxi tip preparation)with 10 ml QBT buffer. QF elution buffer was equilibrated at 50° C. Thesamples were vortexed for 30 seconds, then loaded onto equilibrated tipsand drained by gravity. The tips were washed with 2×15 ml QC buffer. TheDNA was eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with15 ml QF buffer (50° C.). Isopropanol (10.5 ml) was added to eachsample, the tubes covered with parafin and mixed by repeated inversionuntil the DNA precipitated. Samples were pelleted by centrifugation inthe SS-34 rotor at 15,000 rpm for 10 minutes at 4° C. The pelletlocation was marked, the supernatant discarded, and 10 ml 70% ethanol(4° C.) was added. Samples were pelleted again by centrifugation on theSS-34 rotor at 10,000 rpm for 10 minutes at 4° C. The pellet locationwas marked and the supernatant discarded. The tubes were then placed ontheir side in a drying rack and dried 10 minutes at 37° C., taking carenot to overdry the samples.

[0650] After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5)and placed at 50° C. for 1-2 hours. Samples were held overnight at 4° C.as dissolution continued. The DNA solution was then transferred to 1.5ml tubes with a 26 gauge needle on a tuberculin syringe. The transferwas repeated 5× in order to shear the DNA. Samples were then placed at50° C. for 1-2 hours.

[0651] (2) Quantitation of genomic DNA and preparation for geneamplification assay:

[0652] The DNA levels in each tube were quantified by standard A₂₆₀,A₂₈₀ spectrophotometry on a 1:20 dilution (5 μl DNA+95 μl ddH₂O) usingthe 0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer.A₂₆₀/A₂₈₀ ratios were in the range of 1.8-1.9. Each DNA samples was thendiluted further to approximately 200 ng/ml in TE (pH 8.5). If theoriginal material was highly concentrated (about 700 ng/μl ), thematerial was placed at 50° C. for several hours until resuspended.

[0653] Fluorometric DNA quantitation was then performed on the dilutedmaterial (20-600 ng/ml) using the manufacturer's guidelines as modifiedbelow. This was accomplished by allowing a Hoeffer DyNA Quant 200fluorometer to warm-up for about 15 minutes. The Hoechst dye workingsolution (#H33258, 10 μl, prepared within 12 hours of use) was dilutedinto 100 ml 1×TNE buffer. A 2 ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed. pGEM3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solutionand calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA wasthen tested and the reading confirmed at 400 +/−10 units. Each samplewas then read at least in triplicate. When 3 samples were found to bewithin 10% of each other, their average was taken and this value wasused as the quantification value.

[0654] The fluorometricly determined concentration was then used todilute each sample to 10 ng/μl in ddH₂O. This was done simultaneously onall template samples for a single TaqMan plate assay, and with enoughmaterial to run 500-1000 assays. The samples were tested in triplicatewith Taqman™ primers and probe both B-actin and GAPDH on a single platewith normal human DNA and no-template controls. The diluted samples wereused provided that the CT value of normal human DNA subtracted from testDNA was +/−1 Ct. The diluted, lot-qualified genomic DNA was stored in1.0 ml aliquots at −80° C. Aliquots which were subsequently to be usedin the gene amplification assay were stored at 4° C. Each 1 ml aliquotis enough for 8-9 plates or 64 tests

[0655] Gene amplification assay:

[0656] The PRO1800, PRO539, PRO3434 and PRO1927 compounds of theinvention were screened in the following primary tumors and theresulting ΔCt values greater than or equal to 1.0 are reported in Table7 below. TABLE 7 (ΔCt values in lung and colon primary tumor models)Primary Tumor PRO1800 PRO539 PRO3434 PRO1927 LT11 1.65, 1.59, 1.03 LT121.34, 2.28, 1.25 2.03 LT13 1.27, 2.18 1.64, 1.08 5.24, 4.47 4.38 4.80LT15 1.70, 2.23, 1.78, 1.10 1.24 1.00 1.93 LT16 1.00, 1.05, 3.65, 3.192.73, 2.74 1.09 LT17 1.94, 1.63 1.94, 1.01 LT18 1.12 LT19 2.51, 2.181.16 LT21 1.30 1.32 CT2 1.50 CT3 1.17 CT10 1.16 CT12 1.19 CT14 1.62 CT151.48, 1.08 1.03 1.19, 1.40 1.10, 1.30 CT5 1.10 CT11 1.20 1.12 Colo-3201.16 1.78, 1.76, 1.51 (colon tumor cell line) 1.74 HF-00084 2.20 2.41(lung tumor cell line) HCT-116 2.15, 2.22 1.41, 1.47 (colon tumor cellline) HF-00129 1.00, 1.17, 2.31, 5.14 (lung tumor cell line) 4.64 2.401.11 SW-620 1.30 (colon tumor cell line) HT-29 1.64 (colon tumor cellline) SW-403 1.75 (colon tumor cell line) LS174T 1.42 (colon tumor cellline) HCC-2998 1.15 (colon tumor cell line) A549 1.51, 1.09 (lung tumorcell line) Calu-6 1.60, 1.22 (lung tumor cell line) H157 1.61 (lungtumor cell line) H441 1.07, 1.15 (lung tumor cell line) H460 1.01 (lungtumor cell line) SKMES1 1.02 (lung tumor cell line) H810 1.20, 1.54(lung tumor cell line)

Example 17

[0657] Induction of Pancreatic β-Cell Precursor Proliferation (Assay117)

[0658] This assay shows that certain polypeptides of the invention actto induce an increase in the number of pancreatic β-cell precursor cellsand, therefore, are useful for treating various insulin deficient statesin mammals, including diabetes mellitus. The assay is performed asfollows. The assay uses a primary culture of mouse fetal pancreaticcells and the primary readout is an alteration in the expression ofmarkers that represent either β-cell precursors or mature β-cells.Marker expression is measured by real time quantitative PCR (RTQ-PCR);wherein the marker being evaluated is a transcription factor calledPdx1.

[0659] The pancreata are dissected from E14 embryos (CD1 mice). Thepancreata are then digested with collagenase/dispase in F12/DMEM at 37°C. for 40 to 60 minutes (collagenase/dispase, 1.37 mg/ml, BoehringerMannheim, #1097113). The digestion is then neutralized with an equalvolume of 5% BSA and the cells are washed once with RPMI1640. At day 1,the cells are seeded into 12-well tissue culture plates (pre-coated withlaminin, 20 μg/ml in PBS, Boehringer Mannheim, #124317). Cells frompancreata from 1-2 embryos are distributed per well. The culture mediumfor this primary cuture is 14F/1640. At day 2, the media is removed andthe attached cells washed with RPMI/1640. Two mls of minimal media areadded in addition to the protein to be tested. At day 4, the media isremoved and RNA prepared from the cells and marker expression analyzedby real time quantitative RT-PCR. A protein is considered to be activein the assay if it increases the expression of the relevant β-cellmarker as compared to untreated controls.

[0660] 14F/1640 is RPMI1640 (Gibco) plus the following:

[0661] group A 1:1000

[0662] group B 1:1000

[0663] recombinant human insulin 10 μg/ml

[0664] Aprotinin (50 μg/ml) 1:2000 (Boehringer manheim #981532)

[0665] Bovine pituitary extract (BPE) 60 μg/ml

[0666] Gentamycin 100 ng/ml

[0667] Group A : (in 10 ml PBS)

[0668] Transferrin, 100 mg (Sigma T2252)

[0669] Epidermal Growth Factor, 100 μg (BRL 100004)

[0670] Triiodothyronine, 10 μl of 5×10⁻⁶ M (Sigma T5516)

[0671] Ethanolamine, 100 μl of 10⁻¹ M (Sigma E0135)

[0672] Phosphoethalamine, 100 μl of 10⁻¹ M (Sigma P0503)

[0673] Selenium, 4 μl of 10⁻¹ M (Aesar #12574)

[0674] Group C: (in 10 ml 100% ethanol)

[0675] Hydrocortisone, 2 μl of 5×10⁻³ M (Sigma #H0135)

[0676] Progesterone, 100 μl of 1×10⁻³ M (Sigma #P6149)

[0677] Forskolin, 500 μl of 20 mM (Calbiochem #344270)

[0678] Minimal media:

[0679] RPMI 1640 plus transferrin(10 μg/ml), insulin(1 μg/ml),gentamycin(100 ng/ml), aprotinin(50 μg/ml) and BPE (15 μg/ml).

[0680] Defined media:

[0681] RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml),gentamycin (100 ng/ml) and aprotinin (50 μg/ml).

[0682] The following polypeptide was positive in this assay: PRO1868.

Example 18

[0683] Induction of Pancreatic β-Cell Precursor Differentiation (Assay89)

[0684] This assay shows that certain polypeptides of the invention actto induce differentiation of pancreatic β-cell precursor cells intomature pancreatic β-cells and, therefore, are useful for treatingvarious insulin deficient states in mammals, including diabetesmellitus. The assay is performed as follows. The assay uses a primaryculture of mouse fetal pancreatic cells and the primary readout is analteration in the expression of markers that represent either β-cellprecursors or mature β-cells. Marker expression is measured by real timequantitative PCR (RTQ-PCR); wherein the marker being evaluated isinsulin.

[0685] The pancreata are dissected from E14 embryos (CD1 mice). Thepancreata are then digested with collagenase/dispase in F12/DMEM at 37°C. for 40 to 60 minutes (collagenase/dispase, 1.37 mg/ml, BoehringerMannheim, #1097113). The digestion is then neutralized with an equalvolume of 5% BSA and the cells are washed once with RPMI 1640. At day 1,the cells are seeded into 12-well tissue culture plates (pre-coated withlaminin, 20 μg/ml in PBS, Boehringer Mannheim, #124317). Cells frompancreata from 1-2 embryos are distributed per well. The culture mediumfor this primary cuture is 14F/1640. At day 2, the media is removed andthe attached cells washed with RPMI/1640. Two mls of minimal media areadded in addition to the protein to be tested. At day 4, the media isremoved and RNA prepared from the cells and marker expression analyzedby real time quantitative RT-PCR. A protein is considered to be activein the assay if it increases the expression of the relevant β-cellmarker as compared to untreated controls.

[0686] 14F/1640 is RPM11640 (Gibco) plus the following:

[0687] group A 1:1000

[0688] group B 1:1000

[0689] recombinant human insulin 10 μg/ml

[0690] Aprotinin (50 μg/ml) 1:2000 (Boehringer manheim #981532)

[0691] Bovine pituitary extract (BPE) 60 μg/ml

[0692] Gentamycin 100 ng/ml

[0693] Group A: (in 10 ml PBS)

[0694] Transferrin, 100 mg (Sigma T2252)

[0695] Epidermal Growth Factor, 100 μg (BRL 100004)

[0696] Triiodothyronine,10 μl of 5×10⁻⁶ M (Sigma T5516)

[0697] Ethanolamine, 100 μl of 10⁻¹ M (Sigma E0135)

[0698] Phosphoethalamine, 100 μl of 10⁻¹ M (Sigma P0503)

[0699] Selenium, 4 μl of 10⁻¹ M (Aesar #12574)

[0700] Group C : (in 10 ml 100% ethanol)

[0701] Hydrocortisone, 2 μl of 5×10⁻³ M (Sigma #H0135)

[0702] Progesterone, 100 μl of 1×10⁻³ M (Sigma #P6149)

[0703] Forskolin, 500 μl of 20 mM (Calbiochem #344270)

[0704] Minimal media:

[0705] RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml),gentamycin (100 ng/ml), aprotinin(50 μl) and BPE (15 μg/ml).

[0706] Defined media:

[0707] RPMI 1640 plus transferrin (10 μg/ml), insulin (1 μg/ml),gentamycin (100 ng/ml) and aprotinin (50 μg/ml).

[0708] The following polypeptide was positive in this assay: PRO1863.

Example 19

[0709] Mouse Kidney Mesangial Cell Proliferation Assay (Assay 92)

[0710] This assay shows that certain polypeptides of the invention actto induce proliferation of mammalian kidney mesangial cells and,therefore, are useful for treating kidney disorders associated withdecreased mesangial cell function such as Berger disease or othernephropathies associated with Schönlein-Henoch purpura, celiac disease,dermatitis herpetiformis or Crohn disease. The assay is performed asfollows. On day one, mouse kidney mesangial cells are plated on a 96well plate in growth media (3:1 mixture of Dulbecco's modified Eagle'smedium and Ham's F12 medium, 95% fetal bovine serum, 5% supplementedwith 14 mM HEPES) and grown overnight. On day 2, PRO polypeptides arediluted at 2 concentrations(1% and 0.1%) in serum-free medium and addedto the cells. Control samples are serum-free medium alone. On day 4, 20μl of the Cell Titer 96 Aqueous one solution reagent (Progema) was addedto each well and the colormetric reaction was allowed to proceed for 2hours. The absorbance (OD) is then measured at 490 nm. A positive in theassay is anything that gives an absorbance reading which is at least 15%above the control reading.

[0711] The following polypeptide tested positive in this assay: PRO1917.

Example 20

[0712] Fibroblast (BHK-21) Proliferation (Assay 98)

[0713] This assay shows that certain polypeptides of the invention actto induce proliferation of mammalian fibroblast cells in culture and,therefore, function as useful growth factors in mammalian systems. Theassay is performed as follows. BHK-21 fibroblast cells plated instandard growth medium at 2500 cells/well in a total volume of 100 μl.The PRO polypeptide, β-FGF (positive control) or nothing (negativecontrol) are then added to the wells in the presence of 1 μg/ml ofheparin for a total final volume of 200 μl. The cells are then incubatedat 37° C. for 6 to 7 days. After incubation, the media is removed, thecells are washed with PBS and then an acid phosphatase substratereaction mixture (100 μl/well) is added. The cells are then incubated at37° C. for 2 hours. 10 μl per well of 1N NaOH is then added to stop theacid phosphatase reaction. The plates are then read at OD 405 nm. Apositive in the assay is acid phosphatase activity which is at least 50%above the negative control.

[0714] The following polypeptide tested positive in this assay: PRO982.

Example 21

[0715] Chondrocyte Re-differentiation Assay (Assay 110)

[0716] This assay shows that certain polypeptides of the invention actto induce redifferentiation of chondrocytes, therefore, are expected tobe useful for the treatment of various bone and/or cartilage disorderssuch as, for example, sports injuries and arthritis. The assay isperformed as follows. Porcine chondrocytes are isolated by overnightcollagenase digestion of articulary cartilage of metacarpophalangealjoints of 4-6 month old female pigs. The isolated cells are then seededat 25,000 cells/cm² in Ham F-12 containing 10% FBS and 4 μg/mlgentamycin. The culture media is changed every third day and the cellsare then seeded in 96 well plates at 5,000 cells/well in 100 μl of thesame media without serum and 100 μl of the test PRO polypeptide, 5 nMstaurosporin (positive control) or medium alone (negative control) isadded to give a final volume of 200 μl/well. After 5 days of incubationat 37° C., a picture of each well is taken and the differentiation stateof the chondrocytes is determined. A positive result in the assay occurswhen the redifferentiation of the chondrocytes is determined to be moresimilar to the positive control than the negative control.

[0717] The following polypeptide tested positive in this assay: PRO1863.

Example 22

[0718] Use of PRO as a Hybridization Probe

[0719] The following method describes use of a nucleotide sequenceencoding PRO as a hybridization probe.

[0720] DNA comprising the coding sequence of full-length or mature PROas disclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO) in humantissue cDNA libraries or human tissue genomic libraries.

[0721] Hybridization and washing of filters containing either libraryDNAs is performed under the following high stringency conditions.Hybridization of radiolabeled PRO-derived probe to the filters isperformed in a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodiumpyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution,and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filtersis performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

[0722] DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO can then be identified using standardtechniques known in the art.

Example 23

[0723] Expression of PRO in E. Coli

[0724] This example illustrates preparation of an unglycosylated form ofPRO by recombinant expression in E. coli.

[0725] The DNA sequence encoding PRO is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the PRO coding region, lambdatranscriptional terminator, and an argU gene.

[0726] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., suMra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0727] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0728] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

[0729] PRO may be expressed in E. coli in a poly-His tagged form, usingthe following procedure. The DNA encoding PRO is initially amplifiedusing selected PCR primers. The primers will contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures arethen diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate·2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

[0730]E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

[0731] The proteins are refolded by diluting the sample slowly intofreshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA.Refolding volumes are chosen so that the final protein concentration isbetween 50 to 100 micrograms/ml. The refolding solution is stirredgently at 4° C. for 12-36 hours. The refolding reaction is quenched bythe addition of TFA to a final concentration of 0.4% (pH ofapproximately 3). Before further purification of the protein, thesolution is filtered through a 0.22 micron filter and acetonitrile isadded to 2-10% final concentration. The refolded protein ischromatographed on a Poros R1/H reversed phase column using a mobilebuffer of 0.1% TFA with elution with a gradient of acetonitrile from 10to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDSpolyacrylamide gels and fractions containing homogeneous refoldedprotein are pooled. Generally, the properly refolded species of mostproteins are eluted at the lowest concentrations of acetonitrile sincethose species are the most compact with their hydrophobic interiorsshielded from interaction with the reversed phase resin. Aggregatedspecies are usually eluted at higher acetonitrile concentrations. Inaddition to resolving misfolded forms of proteins from the desired form,the reversed phase step also removes endotoxin from the samples.

[0732] Fractions containing the desired folded PRO polypeptide arepooled and the acetonitrile removed using a gentle stream of nitrogendirected at the solution. Proteins are formulated into 20 mM Hepes, pH6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gelfiltration using G25 Superfine (Pharmacia) resins equilibrated in theformulation buffer and sterile filtered.

[0733] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 24

[0734] Expression of PRO in Mammalian Cells

[0735] This example illustrates preparation of a potentiallyglycosylated form of PRO by recombinant expression in mammalian cells.

[0736] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the PRO DNA is ligatedinto pRK5 with selected restriction enzymes to allow insertion of thePRO DNA using ligation methods such as described in Sambrook et al.,supra. The resulting vector is called pRK5-PRO.

[0737] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

[0738] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of PRO polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

[0739] In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

[0740] In another embodiment, PRO can be expressed in CHO cells. ThepRK5-PRO can be transfected into CHO cells using known reagents such asCaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of PRO polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO can then be concentrated and purified byany selected method.

[0741] Epitope-tagged PRO may also be expressed in host CHO cells. ThePRO may be subcloned out of the pRK5 vector. The subclone insert canundergo PCR to fuse in frame with a selected epitope tag such as apoly-his tag into a Baculovirus expression vector. The poly-his taggedPRO insert can then be subcloned into a SV40 driven vector containing aselection marker such as DHFR for selection of stable clones. Finally,the CHO cells can be transfected (as described above) with the SV40driven vector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedPRO can then be concentrated and purified by any selected method, suchas by Ni²⁺-chelate affinity chromatography.

[0742] PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

[0743] Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

[0744] Following PCR amplification, the respective DNAs are subcloned ina CHO expression vector using standard techniques as described inAusubel et al., Current Protocols of Molecular Biology, Unit 3.16, JohnWiley and Sons (1997). CHO expression vectors are constructed to havecompatible restriction sites 5′ and 3′ of the DNA of interest to allowthe convenient shuttling of cDNA's. The vector used expression in CHOcells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

[0745] Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

[0746] The ampules containing the plasmid DNA are thawed by placementinto water bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

[0747] For the poly-His tagged constructs, the proteins are purifiedusing a Ni-NTA column (Qiagen). Before purification, imidazole is addedto the conditioned media to a concentration of 5 mM. The conditionedmedia is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes,pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rateof 4-5 ml/min. at 4° C. After loading, the column is washed withadditional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein is subsequently desalted into a storage buffer containing 10 mMHepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine(Pharmacia) column and stored at −80° C.

[0748] Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

[0749] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 25

[0750] Expression of PRO in Yeast

[0751] The following method describes recombinant expression of PRO inyeast.

[0752] First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

[0753] Yeast cells, such as yeast strain AB110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0754] Recombinant PRO can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

[0755] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 26

[0756] Expression of PRO in Baculovirus-Infected Insect Cells

[0757] The following method describes recombinant expression of PRO inBaculovirus-infected insect cells.

[0758] The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

[0759] Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

[0760] Expressed poly-his tagged PRO can then be purified, for example,by Ni²⁺-chelate affinity chromatography as follows. Extracts areprepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO are pooled and dialyzed againstloading buffer.

[0761] Alternatively, purification of the IgG tagged (or Fc tagged) PROcan be performed using known chromatography techniques, including forinstance, Protein A or protein G column chromatography.

[0762] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 27

[0763] Preparation of Antibodies that Bind PRO

[0764] This example illustrates preparation of monoclonal antibodieswhich can specifically bind PRO.

[0765] Techniques for producing the monoclonal antibodies are known inthe art and are described, for instance, in Goding, supra. Immunogensthat may be employed include purified PRO, fusion proteins containingPRO, and cells expressing recombinant PRO on the cell surface. Selectionof the immunogen can be made by the skilled artisan without undueexperimentation.

[0766] Mice, such as Balb/c, are immunized with the PRO immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

[0767] After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3×63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

[0768] The hybridoma cells will be screened in an ELISA for reactivityagainst PRO. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against PRO is within the skill in theart.

[0769] The positive hybridoma cells can be injected intraperitoneallyinto syngeneic Balb/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 28

[0770] Purification of PRO Polypeptides Using Specific Antibodies

[0771] Native or recombinant PRO polypeptides may be purified by avariety of standard techniques in the art of protein purification. Forexample, pro-PRO polypeptide, mature PRO polypeptide, or pre-PROpolypeptide is purified by immunoaffinity chromatography usingantibodies specific for the PRO polypeptide of interest. In general, animmunoaffinity column is constructed by covalently coupling the anti-PROpolypeptide antibody to an activated chromatographic resin.

[0772] Polyclonal immunoglobulins are prepared from immune sera eitherby precipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

[0773] Such an immunoaffinity column is utilized in the purification ofPRO polypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

[0774] A soluble PRO polypeptide-containing preparation is passed overthe immunoaffinity column, and the column is washed under conditionsthat allow the preferential absorbance of PRO polypeptide (e.g., highionic strength buffers in the presence of detergent). Then, the columnis eluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 29

[0775] Drug Screening

[0776] This invention is particularly useful for screening compounds byusing PRO polypeptides or binding fragment thereof in any of a varietyof drug screening techniques. The PRO polypeptide or fragment employedin such a test may either be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. One methodof drug screening utilizes eukaryotic or prokaryotic host cells whichare stably transformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

[0777] Thus, the present invention provides methods of screening fordrugs or any other agents which can affect a PRO polypeptide-associateddisease or disorder. These methods comprise contacting such an agentwith an PRO polypeptide or fragment thereof and assaying (I) for thepresence of a complex between the agent and the PRO polypeptide orfragment, or (ii) for the presence of a complex between the PROpolypeptide or fragment and the cell, by methods well known in the art.In such competitive binding assays, the PRO polypeptide or fragment istypically labeled. After suitable incubation, free PRO polypeptide orfragment is separated from that present in bound form, and the amount offree or uncomplexed label is a measure of the ability of the particularagent to bind to PRO polypeptide or to interfere with the PROpolypeptide/cell complex.

[0778] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to apolypeptide and is described in detail in WO 84/03564, published on Sep.13, 1984. Briefly stated, large numbers of different small peptide testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. As applied to a PRO polypeptide, the peptide testcompounds are reacted with PRO polypeptide and washed. Bound PROpolypeptide is detected by methods well known in the art. Purified PROpolypeptide can also be coated directly onto plates for use in theaforementioned drug screening techniques. In addition, non-neutralizingantibodies can be used to capture the peptide and immobilize it on thesolid support.

[0779] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 30

[0780] Rational Drug Design

[0781] The goal of rational drug design is to produce structural analogsof biologically active polypeptide of interest (i.e., a PRO polypeptide)or of small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (cf., Hodgson, Bio/Technology, 9: 19-21 (1991)).

[0782] In one approach, the three-dimensional structure of the PROpolypeptide, or of an PRO polypeptide-inhibitor complex, is determinedby x-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).

[0783] It is also possible to isolate a target-specific antibody,selected by functional assay, as described above, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids would be expected to be an analog of the original receptor. Theanti-id could then be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptideswould then act as the pharmacore.

[0784] By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

[0785] Deposit of Material

[0786] The following materials have been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC): Material ATCC Dep. No. Deposit Date DNA35672-2508 203538December 15, 1998 DNA47465-1561 203661 February 9, 1999 DNA57700-1408203583 January 12, 1999 DNA68818-2536 203657 February 9, 1999DNA59847-2510 203576 January 12, 1999 DNA76400-2528 203573 January 12,1999 DNA77624-2515 203553 December 22, 1998 DNA77631-2537 203651Fevruary 9, 1999 DNA82307-2531 203537 December 15, 1998

[0787] These deposit were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposit for 30 years from the date of deposit. The deposits will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Genentech, Inc. and ATCC, which assures permanentand unrestricted availability of the progeny of the culture of thedeposit to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 USC §122 and the Commissioner's rulespursuant thereto (including 37 CFR §1.14 with particular reference to886 OG 638).

[0788] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

[0789] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1 38 1 1283 DNA Homo sapiens 1 cggacgcgtg ggacccatac ttgctggtctgatccatgca caaggcgggg 50 ctgctaggcc tctgtgcccg ggcttggaat tcggtgcggatggccagctc 100 cgggatgacc cgccgggacc cgctcgcaaa taaggtggcc ctggtaacgg150 cctccaccga cgggatcggc ttcgccatcg cccggcgttt ggcccaggac 200ggggcccatg tggtcgtcag cagccggaag cagcagaatg tggaccaggc 250 ggtggccacgctgcaggggg aggggctgag cgtgacgggc accgtgtgcc 300 atgtggggaa ggcggaggaccgggagcggc tggtggccac ggctgtgaag 350 cttcatggag gtatcgatat cctagtctccaatgctgctg tcaacccttt 400 ctttggaagc ataatggatg tcactgagga ggtgtgggacaagactctgg 450 acattaatgt gaaggcccca gccctgatga caaaggcagt ggtgccagaa500 atggagaaac gaggaggcgg ctcagtggtg atcgtgtctt ccatagcagc 550cttcagtcca tctcctggct tcagtcctta caatgtcagt aaaacagcct 600 tgctgggcctgaccaagacc ctggccatag agctggcccc aaggaacatt 650 agggtgaact gcctagcacctggacttatc aagactagct tcagcaggat 700 gctctggatg gacaaggaaa aagaggaaagcatgaaagaa accctgcgga 750 taagaaggtt aggcgagcca gaggattgtg ctggcatcgtgtctttcctg 800 tgctctgaag atgccagcta catcactggg gaaacagtgg tggtgggtgg850 aggaaccccg tcccgcctct gaggaccggg agacagccca caggccagag 900ttgggctcta gctcctggtg ctgttcctgc attcacccac tggcctttcc 950 cacctctgctcaccttactg ttcacctcat caaatcagtt ctgccctgtg 1000 aaaagatcca gccttccctgccgtcaaggt ggcgtcttac tcgggattcc 1050 tgctgttgtt gtggccttgg gtaaaggcctcccctgagaa cacaggacag 1100 gcctgctgac aaggctgagt ctaccttggc aaagaccaagatattttttc 1150 ctgggccact ggtgaatctg aggggtgatg ggagagaagg aacctggagt1200 ggaaggagca gagttgcaaa ttaacagctt gcaaatgagg tgcaaataaa 1250atgcagatga ttgcgcggct ttgaaaaaaa aaa 1283 2 278 PRT Homo sapiens 2 MetHis Lys Ala Gly Leu Leu Gly Leu Cys Ala Arg Ala Trp Asn 1 5 10 15 SerVal Arg Met Ala Ser Ser Gly Met Thr Arg Arg Asp Pro Leu 20 25 30 Ala AsnLys Val Ala Leu Val Thr Ala Ser Thr Asp Gly Ile Gly 35 40 45 Phe Ala IleAla Arg Arg Leu Ala Gln Asp Gly Ala His Val Val 50 55 60 Val Ser Ser ArgLys Gln Gln Asn Val Asp Gln Ala Val Ala Thr 65 70 75 Leu Gln Gly Glu GlyLeu Ser Val Thr Gly Thr Val Cys His Val 80 85 90 Gly Lys Ala Glu Asp ArgGlu Arg Leu Val Ala Thr Ala Val Lys 95 100 105 Leu His Gly Gly Ile AspIle Leu Val Ser Asn Ala Ala Val Asn 110 115 120 Pro Phe Phe Gly Ser IleMet Asp Val Thr Glu Glu Val Trp Asp 125 130 135 Lys Thr Leu Asp Ile AsnVal Lys Ala Pro Ala Leu Met Thr Lys 140 145 150 Ala Val Val Pro Glu MetGlu Lys Arg Gly Gly Gly Ser Val Val 155 160 165 Ile Val Ser Ser Ile AlaAla Phe Ser Pro Ser Pro Gly Phe Ser 170 175 180 Pro Tyr Asn Val Ser LysThr Ala Leu Leu Gly Leu Thr Lys Thr 185 190 195 Leu Ala Ile Glu Leu AlaPro Arg Asn Ile Arg Val Asn Cys Leu 200 205 210 Ala Pro Gly Leu Ile LysThr Ser Phe Ser Arg Met Leu Trp Met 215 220 225 Asp Lys Glu Lys Glu GluSer Met Lys Glu Thr Leu Arg Ile Arg 230 235 240 Arg Leu Gly Glu Pro GluAsp Cys Ala Gly Ile Val Ser Phe Leu 245 250 255 Cys Ser Glu Asp Ala SerTyr Ile Thr Gly Glu Thr Val Val Val 260 265 270 Gly Gly Gly Thr Pro SerArg Leu 275 3 21 DNA Artificial Sequence Synthetic Oligonucleotide Probe3 gcataatgga tgtcactgag g 21 4 23 DNA Artificial Sequence SyntheticOligonucleotide Probe 4 agaacaatcc tgctgaaagc tag 23 5 46 DNA ArtificialSequence Synthetic Oligonucleotide Probe 5 gaaacgagga ggcggctcagtggtgatcgt gtcttccata gcagcc 46 6 3121 DNA Homo sapiens 6 gcgccctgagctccgcctcc gggcccgata gcggcatcga gagcgcctcc 50 gtcgaggacc aggcggcgcagggggccggc gggcgaaagg aggatgaggg 100 ggcgcagcag ctgctgaccc tgcagaaccaggtggcgcgg ctggaggagg 150 agaaccgaga ctttctggct gcgctggagg acgccatggagcagtacaaa 200 ctgcagagcg accggctgcg tgagcagcag gaggagatgg tggaactgcg250 gctgcggtta gagctggtgc ggccaggctg ggggggcctg cggctcctga 300atggcctgcc tcccgggtcc tttgtgcctc gacctcatac agcccccctg 350 gggggtgcccacgcccatgt gctgggcatg gtgccgcctg cctgcctccc 400 tggagatgaa gttggctctgagcagagggg agagcaggtg acaaatggca 450 gggaggctgg agctgagttg ctgactgaggtgaacaggct gggaagtggc 500 tcttcagctg cttcagagga ggaagaggag gaggaggagccgcccaggcg 550 gaccttacac ctgcgcagaa ataggatcag caactgcagt cagagggcgg600 gggcacgccc agggagtctg ccagagagga agggcccaga gctttgcctt 650gaggagttgg atgcagccat tccagggtcc agagcagttg gtgggagcaa 700 ggcccgagttcaggcccgcc aggtcccccc tgccacagcc tcagagtggc 750 ggctggccca ggcccagcagaagatccggg agctggctat caacatccgc 800 atgaaggagg agcttattgg cgagctggtccgcacaggaa aggcagctca 850 ggccctgaac cgccagcaca gccagcgtat ccgggagctggagcaggagg 900 cagagcaggt gcgggccgag ctgagtgaag gccagaggca gctgcgggag950 ctcgagggca aggagctcca ggatgctggc gagcggtctc ggctccagga 1000gttccgcagg agggtcgctg cggcccagag ccaggtgcag gtgctgaagg 1050 agaagaagcaggctacggag cggctggtgt cactgtcggc ccagagtgag 1100 aagcgactgc aggagctcgagcggaacgtg cagctcatgc ggcagcagca 1150 gggacagctg cagaggcggc ttcgcgaggagacggagcag aagcggcgcc 1200 tggaggcaga aatgagcaag cggcagcacc gcgtcaaggagctggagctg 1250 aagcatgagc aacagcagaa gatcctgaag attaagacgg aagagatcgc1300 ggccttccag aggaagaggc gcagtggcag caacggctct gtggtcagcc 1350tggaacagca gcagaagatt gaggagcaga agaagtggct ggaccaggag 1400 atggagaaggtgctacagca gcggcgggcg ctggaggagc tgggggagga 1450 gctccacaag cgggaggccatcctggccaa gaaggaggcc ctgatgcagg 1500 agaagacggg gctggagagc aagcgcctgagatccagcca ggccctcaac 1550 gaggacatcg tgcgagtgtc cagccggctg gagcacctggagaaggagct 1600 gtccgagaag agcgggcagc tgcggcaggg cagcgcccag agccagcagc1650 agatccgcgg ggagatcgac agcctgcgcc aggagaagga ctcgctgctc 1700aagcagcgcc tggagatcga cggcaagctg aggcagggga gtctgctgtc 1750 ccccgaggaggagcggacgc tgttccagtt ggatgaggcc atcgaggccc 1800 tggatgctgc cattgagtataagaatgagg ccatcacatg ccgccagcgg 1850 gtgcttcggg cctcagcctc gttgctgtcccagtgcgaga tgaacctcat 1900 ggccaagctc agctacctct catcctcaga gaccagagccctcctctgca 1950 agtattttga caaggtggtg acgctccgag aggagcagca ccagcagcag2000 attgccttct cggaactgga gatgcagctg gaggagcagc agaggctggt 2050gtactggctg gaggtggccc tggagcggca gcgcctggag atggaccgcc 2100 agctgaccctgcagcagaag gagcacgagc agaacatgca gctgctcctg 2150 cagcagagtc gagaccacctcggtgaaggg ttagcagaca gcaggaggca 2200 gtatgaggcc cggattcaag ctctggagaaggaactgggc cgttacatgt 2250 ggataaacca ggaactgaaa cagaagctcg gcggtgtgaacgctgtaggc 2300 cacagcaggg gtggggagaa gaggagcctg tgctcggagg gcagacaggc2350 tcctggaaat gaagatgagc tccacctggc acccgagctt ctctggctgt 2400cccccctcac tgagggggcc ccccgcaccc gggaggagac gcgggacttg 2450 gtccacgctccgttaccctt gacctggaaa cgctcgagcc tgtgtggtga 2500 ggagcagggg tcccccgaggaactgaggca gcgggaggcg gctgagcccc 2550 tggtggggcg ggtgcttcct gtgggtgaggcaggcctgcc ctggaacttt 2600 gggcctttgt ccaagccccg gcgggaactg cgacgagccagcccggggat 2650 gattgatgtc cggaaaaacc ccctgtaagc cctcggggca gaccctgcct2700 tggagggaga ctccgagcct gctgaaaggg gcagctgcct gttttgcttc 2750tgtgaagggc agtccttacc gcacacccta aatccaggcc ctcatctgta 2800 ccctcactgggatcaacaaa tttgggccat ggcccaaaag aactggaccc 2850 tcatttaaca aaataatatgcaaattccca ccacttactt ccatgaagct 2900 gtggtaccca attgccgcct tgtgtcttgctcgaatctca ggacaattct 2950 ggtttcaggc gtaaatggat gtgcttgtag ttcaggggtttggccaagaa 3000 tcatcacgaa agggtcggtg gcaaccaggt tgtggtttaa atggtcttat3050 gtatataggg gaaactggga gactttagga tcttaaaaaa ccatttaata 3100aaaaaaaatc tttgaaggga c 3121 7 830 PRT Homo sapiens 7 Met Glu Gln TyrLys Leu Gln Ser Asp Arg Leu Arg Glu Gln Gln 1 5 10 15 Glu Glu Met ValGlu Leu Arg Leu Arg Leu Glu Leu Val Arg Pro 20 25 30 Gly Trp Gly Gly LeuArg Leu Leu Asn Gly Leu Pro Pro Gly Ser 35 40 45 Phe Val Pro Arg Pro HisThr Ala Pro Leu Gly Gly Ala His Ala 50 55 60 His Val Leu Gly Met Val ProPro Ala Cys Leu Pro Gly Asp Glu 65 70 75 Val Gly Ser Glu Gln Arg Gly GluGln Val Thr Asn Gly Arg Glu 80 85 90 Ala Gly Ala Glu Leu Leu Thr Glu ValAsn Arg Leu Gly Ser Gly 95 100 105 Ser Ser Ala Ala Ser Glu Glu Glu GluGlu Glu Glu Glu Pro Pro 110 115 120 Arg Arg Thr Leu His Leu Arg Arg AsnArg Ile Ser Asn Cys Ser 125 130 135 Gln Arg Ala Gly Ala Arg Pro Gly SerLeu Pro Glu Arg Lys Gly 140 145 150 Pro Glu Leu Cys Leu Glu Glu Leu AspAla Ala Ile Pro Gly Ser 155 160 165 Arg Ala Val Gly Gly Ser Lys Ala ArgVal Gln Ala Arg Gln Val 170 175 180 Pro Pro Ala Thr Ala Ser Glu Trp ArgLeu Ala Gln Ala Gln Gln 185 190 195 Lys Ile Arg Glu Leu Ala Ile Asn IleArg Met Lys Glu Glu Leu 200 205 210 Ile Gly Glu Leu Val Arg Thr Gly LysAla Ala Gln Ala Leu Asn 215 220 225 Arg Gln His Ser Gln Arg Ile Arg GluLeu Glu Gln Glu Ala Glu 230 235 240 Gln Val Arg Ala Glu Leu Ser Glu GlyGln Arg Gln Leu Arg Glu 245 250 255 Leu Glu Gly Lys Glu Leu Gln Asp AlaGly Glu Arg Ser Arg Leu 260 265 270 Gln Glu Phe Arg Arg Arg Val Ala AlaAla Gln Ser Gln Val Gln 275 280 285 Val Leu Lys Glu Lys Lys Gln Ala ThrGlu Arg Leu Val Ser Leu 290 295 300 Ser Ala Gln Ser Glu Lys Arg Leu GlnGlu Leu Glu Arg Asn Val 305 310 315 Gln Leu Met Arg Gln Gln Gln Gly GlnLeu Gln Arg Arg Leu Arg 320 325 330 Glu Glu Thr Glu Gln Lys Arg Arg LeuGlu Ala Glu Met Ser Lys 335 340 345 Arg Gln His Arg Val Lys Glu Leu GluLeu Lys His Glu Gln Gln 350 355 360 Gln Lys Ile Leu Lys Ile Lys Thr GluGlu Ile Ala Ala Phe Gln 365 370 375 Arg Lys Arg Arg Ser Gly Ser Asn GlySer Val Val Ser Leu Glu 380 385 390 Gln Gln Gln Lys Ile Glu Glu Gln LysLys Trp Leu Asp Gln Glu 395 400 405 Met Glu Lys Val Leu Gln Gln Arg ArgAla Leu Glu Glu Leu Gly 410 415 420 Glu Glu Leu His Lys Arg Glu Ala IleLeu Ala Lys Lys Glu Ala 425 430 435 Leu Met Gln Glu Lys Thr Gly Leu GluSer Lys Arg Leu Arg Ser 440 445 450 Ser Gln Ala Leu Asn Glu Asp Ile ValArg Val Ser Ser Arg Leu 455 460 465 Glu His Leu Glu Lys Glu Leu Ser GluLys Ser Gly Gln Leu Arg 470 475 480 Gln Gly Ser Ala Gln Ser Gln Gln GlnIle Arg Gly Glu Ile Asp 485 490 495 Ser Leu Arg Gln Glu Lys Asp Ser LeuLeu Lys Gln Arg Leu Glu 500 505 510 Ile Asp Gly Lys Leu Arg Gln Gly SerLeu Leu Ser Pro Glu Glu 515 520 525 Glu Arg Thr Leu Phe Gln Leu Asp GluAla Ile Glu Ala Leu Asp 530 535 540 Ala Ala Ile Glu Tyr Lys Asn Glu AlaIle Thr Cys Arg Gln Arg 545 550 555 Val Leu Arg Ala Ser Ala Ser Leu LeuSer Gln Cys Glu Met Asn 560 565 570 Leu Met Ala Lys Leu Ser Tyr Leu SerSer Ser Glu Thr Arg Ala 575 580 585 Leu Leu Cys Lys Tyr Phe Asp Lys ValVal Thr Leu Arg Glu Glu 590 595 600 Gln His Gln Gln Gln Ile Ala Phe SerGlu Leu Glu Met Gln Leu 605 610 615 Glu Glu Gln Gln Arg Leu Val Tyr TrpLeu Glu Val Ala Leu Glu 620 625 630 Arg Gln Arg Leu Glu Met Asp Arg GlnLeu Thr Leu Gln Gln Lys 635 640 645 Glu His Glu Gln Asn Met Gln Leu LeuLeu Gln Gln Ser Arg Asp 650 655 660 His Leu Gly Glu Gly Leu Ala Asp SerArg Arg Gln Tyr Glu Ala 665 670 675 Arg Ile Gln Ala Leu Glu Lys Glu LeuGly Arg Tyr Met Trp Ile 680 685 690 Asn Gln Glu Leu Lys Gln Lys Leu GlyGly Val Asn Ala Val Gly 695 700 705 His Ser Arg Gly Gly Glu Lys Arg SerLeu Cys Ser Glu Gly Arg 710 715 720 Gln Ala Pro Gly Asn Glu Asp Glu LeuHis Leu Ala Pro Glu Leu 725 730 735 Leu Trp Leu Ser Pro Leu Thr Glu GlyAla Pro Arg Thr Arg Glu 740 745 750 Glu Thr Arg Asp Leu Val His Ala ProLeu Pro Leu Thr Trp Lys 755 760 765 Arg Ser Ser Leu Cys Gly Glu Glu GlnGly Ser Pro Glu Glu Leu 770 775 780 Arg Gln Arg Glu Ala Ala Glu Pro LeuVal Gly Arg Val Leu Pro 785 790 795 Val Gly Glu Ala Gly Leu Pro Trp AsnPhe Gly Pro Leu Ser Lys 800 805 810 Pro Arg Arg Glu Leu Arg Arg Ala SerPro Gly Met Ile Asp Val 815 820 825 Arg Lys Asn Pro Leu 830 8 662 DNAHomo sapiens 8 attctcctag agcatctttg gaagcatgag gccacgatgc tgcatcttgg 50ctcttgtctg ctggataaca gtcttcctcc tccagtgttc aaaaggaact 100 acagacgctcctgttggctc aggactgtgg ctgtgccagc cgacacccag 150 gtgtgggaac aagatctacaacccttcaga gcagtgctgt tatgatgatg 200 ccatcttatc cttaaaggag acccgccgctgtggctccac ctgcaccttc 250 tggccctgct ttgagctctg ctgtcccgag tcttttggcccccagcagaa 300 gtttcttgtg aagttgaggg ttctgggtat gaagtctcag tgtcacttat350 ctcccatctc ccggagctgt accaggaaca ggaggcacgt cctgtaccca 400taaaaacccc aggctccact ggcagacggc agacaagggg agaagagacg 450 aagcagctggacatcggaga ctacagttga acttcggaga gaagcaactt 500 gacttcagag ggatggctcaatgacatagc tttggagagg agcccagctg 550 gggatggcca gacttcaggg gaagaatgccttcctgcttc atcccctttc 600 cagctcccct tcccgctgag agccactttc atcggcaataaaatccccca 650 catttaccat ct 662 9 125 PRT Homo sapiens 9 Met Arg ProArg Cys Cys Ile Leu Ala Leu Val Cys Trp Ile Thr 1 5 10 15 Val Phe LeuLeu Gln Cys Ser Lys Gly Thr Thr Asp Ala Pro Val 20 25 30 Gly Ser Gly LeuTrp Leu Cys Gln Pro Thr Pro Arg Cys Gly Asn 35 40 45 Lys Ile Tyr Asn ProSer Glu Gln Cys Cys Tyr Asp Asp Ala Ile 50 55 60 Leu Ser Leu Lys Glu ThrArg Arg Cys Gly Ser Thr Cys Thr Phe 65 70 75 Trp Pro Cys Phe Glu Leu CysCys Pro Glu Ser Phe Gly Pro Gln 80 85 90 Gln Lys Phe Leu Val Lys Leu ArgVal Leu Gly Met Lys Ser Gln 95 100 105 Cys His Leu Ser Pro Ile Ser ArgSer Cys Thr Arg Asn Arg Arg 110 115 120 His Val Leu Tyr Pro 125 10 1942DNA Homo sapiens 10 cccacgcgtc cgcccacgcg tccgggtgcc actcgcgcgccggccgcgct 50 ccgggcttct cttttccctc cgacgcgcca cggctgccca gacattccgg 100ctgccgggtc tggagagctc cccgaacccc tccgcggaga ggagcgaggc 150 ggcgccagggtggcccccgg ggcgcgcttg gtctcggaga agcggggacg 200 aggccggagg atgagcgactgagggcgacg cgggcactga cgcgagttgg 250 ggccgcgact accggcagct gacagcgcgatgagcgactc cccagagacg 300 ccctagcccg gtgtgcgcgc caggcggagc gcgcaggtggggctgggctg 350 ttagtggtcc gccccacgcg ggtcgccggc cggcccagga tgggcgctgg400 caacccgggc ccgcgcccgc cgctgctacc cctgcgcccg ctgcgagccc 450ggcgtccggc ccgcgccctg cgctcatgga cggcggctcc cggctggcgg 500 cggcgcgcccccgggctgtg aatgcgactc gcccctcggc cgcgctcccc 550 gcccgcccgc ccgccgggacgtggtagggg atgcccagct ccactgcgat 600 ggcagttggc gcgctctcca gttccctcctggtcacctgc tgcctgatgg 650 tggctctgtg cagtccgagc atcccgctgg agaagctggcccaggcacca 700 gagcagccgg gccaggagaa gcgtgagcac gccactcggg acggcccggg750 gcgggtgaac gagctcgggc gcccggcgag ggacgagggc ggcagcggcc 800gggactggaa gagcaagagc ggccgtgggc tcgccggccg tgagccgtgg 850 agcaagctgaagcaggcctg ggtctcccag ggcgggggcg ccaaggccgg 900 ggatctgcag gtccggccccgcggggacac cccgcaggcg gaagccctgg 950 ccgcagccgc ccaggacgcg attggcccggaactcgcgcc cacgcccgag 1000 ccacccgagg agtacgtgta cccggactac cgtggcaagggctgcgtgga 1050 cgagagcggc ttcgtgtacg cgatcgggga gaagttcgcg ccgggcccct1100 cggcctgccc gtgcctgtgc accgaggagg ggccgctgtg cgcgcagccc 1150gagtgcccga ggctgcaccc gcgctgcatc cacgtcgaca cgagccagtg 1200 ctgcccgcagtgcaaggaga ggaagaacta ctgcgagttc cggggcaaga 1250 cctatcagac tttggaggagttcgtggtgt ctccatgcga gaggtgtcgc 1300 tgtgaagcca acggtgaggt gctatgcacagtgtcagcgt gtccccagac 1350 ggagtgtgtg gaccctgtgt acgagcctga tcagtgctgtcccatctgca 1400 aaaatggtcc aaactgcttt gcagaaaccg cggtgatccc tgctggcaga1450 gaagtgaaga ctgacgagtg caccatatgc cactgtactt atgaggaagg 1500cacatggaga atcgagcggc aggccatgtg cacgagacat gaatgcaggc 1550 aaatgtagacgcttcccaga acacaaactc tgactttttc tagaacattt 1600 tactgatgtg aacattctagatgactctgg gaactatcag tcaaagaaga 1650 cttttgatga ggaataatgg aaaattgttggtacttttcc ttttcttgat 1700 aacagttact acaacagaag gaaatggata tatttcaaaacatcaacaag 1750 aactttgggc ataaaatcct tctctaaata aatgtgctat tttcacagta1800 agtacacaaa agtacactat tatatatcaa atgtatttct ataatccctc 1850cattagagag cttatataag tgttttctat agatgcagat taaaaatgct 1900 gtgttgtcaaccgtcaaaaa aaaaaaaaaa aaaaaaaaaa aa 1942 11 325 PRT Homo sapiens 11 MetPro Ser Ser Thr Ala Met Ala Val Gly Ala Leu Ser Ser Ser 1 5 10 15 LeuLeu Val Thr Cys Cys Leu Met Val Ala Leu Cys Ser Pro Ser 20 25 30 Ile ProLeu Glu Lys Leu Ala Gln Ala Pro Glu Gln Pro Gly Gln 35 40 45 Glu Lys ArgGlu His Ala Thr Arg Asp Gly Pro Gly Arg Val Asn 50 55 60 Glu Leu Gly ArgPro Ala Arg Asp Glu Gly Gly Ser Gly Arg Asp 65 70 75 Trp Lys Ser Lys SerGly Arg Gly Leu Ala Gly Arg Glu Pro Trp 80 85 90 Ser Lys Leu Lys Gln AlaTrp Val Ser Gln Gly Gly Gly Ala Lys 95 100 105 Ala Gly Asp Leu Gln ValArg Pro Arg Gly Asp Thr Pro Gln Ala 110 115 120 Glu Ala Leu Ala Ala AlaAla Gln Asp Ala Ile Gly Pro Glu Leu 125 130 135 Ala Pro Thr Pro Glu ProPro Glu Glu Tyr Val Tyr Pro Asp Tyr 140 145 150 Arg Gly Lys Gly Cys ValAsp Glu Ser Gly Phe Val Tyr Ala Ile 155 160 165 Gly Glu Lys Phe Ala ProGly Pro Ser Ala Cys Pro Cys Leu Cys 170 175 180 Thr Glu Glu Gly Pro LeuCys Ala Gln Pro Glu Cys Pro Arg Leu 185 190 195 His Pro Arg Cys Ile HisVal Asp Thr Ser Gln Cys Cys Pro Gln 200 205 210 Cys Lys Glu Arg Lys AsnTyr Cys Glu Phe Arg Gly Lys Thr Tyr 215 220 225 Gln Thr Leu Glu Glu PheVal Val Ser Pro Cys Glu Arg Cys Arg 230 235 240 Cys Glu Ala Asn Gly GluVal Leu Cys Thr Val Ser Ala Cys Pro 245 250 255 Gln Thr Glu Cys Val AspPro Val Tyr Glu Pro Asp Gln Cys Cys 260 265 270 Pro Ile Cys Lys Asn GlyPro Asn Cys Phe Ala Glu Thr Ala Val 275 280 285 Ile Pro Ala Gly Arg GluVal Lys Thr Asp Glu Cys Thr Ile Cys 290 295 300 His Cys Thr Tyr Glu GluGly Thr Trp Arg Ile Glu Arg Gln Ala 305 310 315 Met Cys Thr Arg His GluCys Arg Gln Met 320 325 12 24 DNA Artificial Sequence SyntheticOligonucleotide Probe 12 gaggtgtcgc tgtgaagcca acgg 24 13 24 DNAArtificial Sequence Synthetic Oligonucleotide Probe 13 cgctcgattctccatgtgcc ttcc 24 14 45 DNA Artificial Sequence SyntheticOligonucleotide Probe 14 gacggagtgt gtggaccctg tgtacgagcc tgatcagtgctgtcc 45 15 1587 DNA Homo sapiens 15 cagccacaga cgggtcatga gcgcggtattactgctggcc ctcctggggt 50 tcatcctccc actgccagga gtgcaggcgc tgctctgccagtttgggaca 100 gttcagcatg tgtggaaggt gtccgaccta ccccggcaat ggacccctaa150 gaacaccagc tgcgacagcg gcttggggtg ccaggacacg ttgatgctca 200ttgagagcgg accccaagtg agcctggtgc tctccaaggg ctgcacggag 250 gccaaggaccaggagccccg cgtcactgag caccggatgg gccccggcct 300 ctccctgatc tcctacaccttcgtgtgccg ccaggaggac ttctgcaaca 350 acctcgttaa ctccctcccg ctttgggccccacagccccc agcagaccca 400 ggatccttga ggtgcccagt ctgcttgtct atggaaggctgtctggaggg 450 gacaacagaa gagatctgcc ccaaggggac cacacactgt tatgatggcc500 tcctcaggct caggggagga ggcatcttct ccaatctgag agtccaggga 550tgcatgcccc agccaggttg caacctgctc aatgggacac aggaaattgg 600 gcccgtgggtatgactgaga actgcaatag gaaagatttt ctgacctgtc 650 atcgggggac caccattatgacacacggaa acttggctca agaacccact 700 gattggacca catcgaatac cgagatgtgcgaggtggggc aggtgtgtca 750 ggagacgctg ctgctcatag atgtaggact cacatcaaccctggtgggga 800 caaaaggctg cagcactgtt ggggctcaaa attcccagaa gaccaccatc850 cactcagccc ctcctggggt gcttgtggcc tcctataccc acttctgctc 900ctcggacctg tgcaatagtg ccagcagcag cagcgttctg ctgaactccc 950 tccctcctcaagctgcccct gtcccaggag accggcagtg tcctacctgt 1000 gtgcagcccc ttggaacctgttcaagtggc tccccccgaa tgacctgccc 1050 caggggcgcc actcattgtt atgatgggtacattcatctc tcaggaggtg 1100 ggctgtccac caaaatgagc attcagggct gcgtggcccaaccttccagc 1150 ttcttgttga accacaccag acaaatcggg atcttctctg cgcgtgagaa1200 gcgtgatgtg cagcctcctg cctctcagca tgagggaggt ggggctgagg 1250gcctggagtc tctcacttgg ggggtggggc tggcactggc cccagcgctg 1300 tggtggggagtggtttgccc ttcctgctaa ctctattacc cccacgattc 1350 ttcaccgctg ctgaccacccacactcaacc tccctctgac ctcataacct 1400 aatggccttg gacaccagat tctttcccattctgtccatg aatcatcttc 1450 cccacacaca atcattcata tctactcacc taacagcaacactggggaga 1500 gcctggagca tccggacttg ccctatggga gaggggacgc tggaggagtg1550 gctgcatgta tctgataata cagaccctgt cctttca 1587 16 437 PRT Homosapiens 16 Met Ser Ala Val Leu Leu Leu Ala Leu Leu Gly Phe Ile Leu Pro 15 10 15 Leu Pro Gly Val Gln Ala Leu Leu Cys Gln Phe Gly Thr Val Gln 2025 30 His Val Trp Lys Val Ser Asp Leu Pro Arg Gln Trp Thr Pro Lys 35 4045 Asn Thr Ser Cys Asp Ser Gly Leu Gly Cys Gln Asp Thr Leu Met 50 55 60Leu Ile Glu Ser Gly Pro Gln Val Ser Leu Val Leu Ser Lys Gly 65 70 75 CysThr Glu Ala Lys Asp Gln Glu Pro Arg Val Thr Glu His Arg 80 85 90 Met GlyPro Gly Leu Ser Leu Ile Ser Tyr Thr Phe Val Cys Arg 95 100 105 Gln GluAsp Phe Cys Asn Asn Leu Val Asn Ser Leu Pro Leu Trp 110 115 120 Ala ProGln Pro Pro Ala Asp Pro Gly Ser Leu Arg Cys Pro Val 125 130 135 Cys LeuSer Met Glu Gly Cys Leu Glu Gly Thr Thr Glu Glu Ile 140 145 150 Cys ProLys Gly Thr Thr His Cys Tyr Asp Gly Leu Leu Arg Leu 155 160 165 Arg GlyGly Gly Ile Phe Ser Asn Leu Arg Val Gln Gly Cys Met 170 175 180 Pro GlnPro Gly Cys Asn Leu Leu Asn Gly Thr Gln Glu Ile Gly 185 190 195 Pro ValGly Met Thr Glu Asn Cys Asn Arg Lys Asp Phe Leu Thr 200 205 210 Cys HisArg Gly Thr Thr Ile Met Thr His Gly Asn Leu Ala Gln 215 220 225 Glu ProThr Asp Trp Thr Thr Ser Asn Thr Glu Met Cys Glu Val 230 235 240 Gly GlnVal Cys Gln Glu Thr Leu Leu Leu Ile Asp Val Gly Leu 245 250 255 Thr SerThr Leu Val Gly Thr Lys Gly Cys Ser Thr Val Gly Ala 260 265 270 Gln AsnSer Gln Lys Thr Thr Ile His Ser Ala Pro Pro Gly Val 275 280 285 Leu ValAla Ser Tyr Thr His Phe Cys Ser Ser Asp Leu Cys Asn 290 295 300 Ser AlaSer Ser Ser Ser Val Leu Leu Asn Ser Leu Pro Pro Gln 305 310 315 Ala AlaPro Val Pro Gly Asp Arg Gln Cys Pro Thr Cys Val Gln 320 325 330 Pro LeuGly Thr Cys Ser Ser Gly Ser Pro Arg Met Thr Cys Pro 335 340 345 Arg GlyAla Thr His Cys Tyr Asp Gly Tyr Ile His Leu Ser Gly 350 355 360 Gly GlyLeu Ser Thr Lys Met Ser Ile Gln Gly Cys Val Ala Gln 365 370 375 Pro SerSer Phe Leu Leu Asn His Thr Arg Gln Ile Gly Ile Phe 380 385 390 Ser AlaArg Glu Lys Arg Asp Val Gln Pro Pro Ala Ser Gln His 395 400 405 Glu GlyGly Gly Ala Glu Gly Leu Glu Ser Leu Thr Trp Gly Val 410 415 420 Gly LeuAla Leu Ala Pro Ala Leu Trp Trp Gly Val Val Cys Pro 425 430 435 Ser Cys17 2387 DNA Homo sapiens 17 cgacgatgct acgcgcgccc ggctgcctcc tccggacctccgtagcgcct 50 gccgcggccc tggctgcggc gctgctctcg tcgcttgcgc gctgctctct 100tctagagccg agggacccgg tggcctcgtc gctcagcccc tatttcggca 150 ccaagactcgctacgaggat gtcaaccccg tgctattgtc gggccccgag 200 gctccgtggc gggaccctgagctgctggag gggacctgca ccccggtgca 250 gctggtcgcc ctcattcgcc acggcacccgctaccccacg gtcaaacaga 300 tccgcaagct gaggcagctg cacgggttgc tgcaggcccgcgggtccagg 350 gatggcgggg ctagtagtac cggcagccgc gacctgggtg cagcgctggc400 cgactggcct ttgtggtacg cggactggat ggacgggcag ctagtagaga 450agggacggca ggatatgcga cagctggcgc tgcgtctggc ctcgctcttc 500 ccggcccttttcagccgtga gaactacggc cgcctgcggc tcatcaccag 550 ttccaagcac cgctgcatggatagcagcgc cgccttcctg caggggctgt 600 ggcagcacta ccaccctggc ttgccgccgccggacgtcgc agatatggag 650 tttggacctc caacagttaa tgataaacta atgagattttttgatcactg 700 tgagaagttt ttaactgaag tagaaaaaaa tgctacagct ctttatcacg750 tggaagcctt caaaactgga ccagaaatgc agaacatttt aaaaaaagtt 800gcagctactt tgcaagtgcc agtaaatgat ttaaatgcag atttaattca 850 agtagcctttttcacctgtt catttgacct ggcaattaaa ggtgttaaat 900 ctccttggtg tgatgtttttgacatagatg atgcaaaggt attagaatat 950 ttaaatgatc tgaaacaata ttggaaaagaggatatgggt atactattaa 1000 cagtcgatcc agctgcacct tgtttcagga tatctttcagcacttggaca 1050 aagcagttga acagaaacaa aggtctcagc caatttcttc tccagtcatc1100 ctccagtttg gtcatgcaga gactcttctt ccactgcttt ctctcatggg 1150ctacttcaaa gacaaggaac ccctaacagc gtacaattac aaaaaacaaa 1200 tgcatcggaagttccgaagt ggtctcattg taccttatgc ctcgaacctg 1250 atatttgtgc tttaccactgtgaaaatgct aagactccta aagaacaatt 1300 ccgagtgcag atgttattaa atgaaaaggtgttacctttg gcttactcac 1350 aagaaactgt ttcattttat gaagatctga agaaccactacaaggacatc 1400 cttcagagtt gtcaaaccag tgaagaatgt gaattagcaa gggctaacag1450 tacatctgat gaactatgag taactgaaga acatttttaa ttctttagga 1500atctgcaatg agtgattaca tgcttgtaat aggtaggcaa ttccttgatt 1550 acaggaagcttttatattac ttgagtattt ctgtcttttc acagaaaaac 1600 attgggtttc tctctgggtttggacatgaa atgtaagaaa agatttttca 1650 ctggagcagc tctcttaagg agaaacaaatctatttagag aaacagctgg 1700 ccctgcaaat gtttacagaa atgaaattct tcctacttatataagaaatc 1750 tcacactgag atagaattgt gatttcataa taacacttga aaagtgctgg1800 agtaacaaaa tatctcagtt ggaccatcct taacttgatt gaactgtcta 1850ggaactttac agattgttct gcagttctct cttcttttcc tcaggtagga 1900 cagctctagcattttcttaa tcaggaatat tgtggtaagc tgggagtatc 1950 actctggaag aaagtaacatctccagatga gaatttgaaa caagaaacag 2000 agtgttgtaa aaggacacct tcactgaagcaagtcggaaa gtacaatgaa 2050 aataaatatt tttggtattt atttatgaaa tatttgaacattttttcaat 2100 aattcctttt tacttctagg aagtctcaaa agaccatctt aaattattat2150 atgtttggac aattagcaac aagtcagata gttagaatcg aagtttttca 2200aatccattgc ttagctaact ttttcattct gtcacttggc ttcgattttt 2250 atattttcctattatatgaa atgtatcttt tggttgtttg atttttcttt 2300 ctttctttgt aaatagttctgagttctgtc aaatgccgtg aaagtatttg 2350 ctataataaa gaaaattctt gtgactttaaaaaaaaa 2387 18 487 PRT Homo sapiens 18 Met Leu Arg Ala Pro Gly Cys LeuLeu Arg Thr Ser Val Ala Pro 1 5 10 15 Ala Ala Ala Leu Ala Ala Ala LeuLeu Ser Ser Leu Ala Arg Cys 20 25 30 Ser Leu Leu Glu Pro Arg Asp Pro ValAla Ser Ser Leu Ser Pro 35 40 45 Tyr Phe Gly Thr Lys Thr Arg Tyr Glu AspVal Asn Pro Val Leu 50 55 60 Leu Ser Gly Pro Glu Ala Pro Trp Arg Asp ProGlu Leu Leu Glu 65 70 75 Gly Thr Cys Thr Pro Val Gln Leu Val Ala Leu IleArg His Gly 80 85 90 Thr Arg Tyr Pro Thr Val Lys Gln Ile Arg Lys Leu ArgGln Leu 95 100 105 His Gly Leu Leu Gln Ala Arg Gly Ser Arg Asp Gly GlyAla Ser 110 115 120 Ser Thr Gly Ser Arg Asp Leu Gly Ala Ala Leu Ala AspTrp Pro 125 130 135 Leu Trp Tyr Ala Asp Trp Met Asp Gly Gln Leu Val GluLys Gly 140 145 150 Arg Gln Asp Met Arg Gln Leu Ala Leu Arg Leu Ala SerLeu Phe 155 160 165 Pro Ala Leu Phe Ser Arg Glu Asn Tyr Gly Arg Leu ArgLeu Ile 170 175 180 Thr Ser Ser Lys His Arg Cys Met Asp Ser Ser Ala AlaPhe Leu 185 190 195 Gln Gly Leu Trp Gln His Tyr His Pro Gly Leu Pro ProPro Asp 200 205 210 Val Ala Asp Met Glu Phe Gly Pro Pro Thr Val Asn AspLys Leu 215 220 225 Met Arg Phe Phe Asp His Cys Glu Lys Phe Leu Thr GluVal Glu 230 235 240 Lys Asn Ala Thr Ala Leu Tyr His Val Glu Ala Phe LysThr Gly 245 250 255 Pro Glu Met Gln Asn Ile Leu Lys Lys Val Ala Ala ThrLeu Gln 260 265 270 Val Pro Val Asn Asp Leu Asn Ala Asp Leu Ile Gln ValAla Phe 275 280 285 Phe Thr Cys Ser Phe Asp Leu Ala Ile Lys Gly Val LysSer Pro 290 295 300 Trp Cys Asp Val Phe Asp Ile Asp Asp Ala Lys Val LeuGlu Tyr 305 310 315 Leu Asn Asp Leu Lys Gln Tyr Trp Lys Arg Gly Tyr GlyTyr Thr 320 325 330 Ile Asn Ser Arg Ser Ser Cys Thr Leu Phe Gln Asp IlePhe Gln 335 340 345 His Leu Asp Lys Ala Val Glu Gln Lys Gln Arg Ser GlnPro Ile 350 355 360 Ser Ser Pro Val Ile Leu Gln Phe Gly His Ala Glu ThrLeu Leu 365 370 375 Pro Leu Leu Ser Leu Met Gly Tyr Phe Lys Asp Lys GluPro Leu 380 385 390 Thr Ala Tyr Asn Tyr Lys Lys Gln Met His Arg Lys PheArg Ser 395 400 405 Gly Leu Ile Val Pro Tyr Ala Ser Asn Leu Ile Phe ValLeu Tyr 410 415 420 His Cys Glu Asn Ala Lys Thr Pro Lys Glu Gln Phe ArgVal Gln 425 430 435 Met Leu Leu Asn Glu Lys Val Leu Pro Leu Ala Tyr SerGln Glu 440 445 450 Thr Val Ser Phe Tyr Glu Asp Leu Lys Asn His Tyr LysAsp Ile 455 460 465 Leu Gln Ser Cys Gln Thr Ser Glu Glu Cys Glu Leu AlaArg Ala 470 475 480 Asn Ser Thr Ser Asp Glu Leu 485 19 3554 DNA Homosapiens 19 gggactacaa gccgcgccgc gctgccgctg gcccctcagc aaccctcgac 50atggcgctga ggcggccacc gcgactccgg ctctgcgctc ggctgcctga 100 cttcttcctgctgctgcttt tcaggggctg cctgataggg gctgtaaatc 150 tcaaatccag caatcgaaccccagtggtac aggaatttga aagtgtggaa 200 ctgtcttgca tcattacgga ttcgcagacaagtgacccca ggatcgagtg 250 gaagaaaatt caagatgaac aaaccacata tgtgttttttgacaacaaaa 300 ttcagggaga cttggcgggt cgtgcagaaa tactggggaa gacatccctg350 aagatctgga atgtgacacg gagagactca gccctttatc gctgtgaggt 400cgttgctcga aatgaccgca aggaaattga tgagattgtg atcgagttaa 450 ctgtgcaagtgaagccagtg acccctgtct gtagagtgcc gaaggctgta 500 ccagtaggca agatggcaacactgcactgc caggagagtg agggccaccc 550 ccggcctcac tacagctggt atcgcaatgatgtaccactg cccacggatt 600 ccagagccaa tcccagattt cgcaattctt ctttccacttaaactctgaa 650 acaggcactt tggtgttcac tgctgttcac aaggacgact ctgggcagta700 ctactgcatt gcttccaatg acgcaggctc agccaggtgt gaggagcagg 750agatggaagt ctatgacctg aacattggcg gaattattgg gggggttctg 800 gttgtccttgctgtactggc cctgatcacg ttgggcatct gctgtgcata 850 cagacgtggc tacttcatcaacaataaaca ggatggagaa agttacaaga 900 acccagggaa accagatgga gttaactacatccgcactga cgaggagggc 950 gacttcagac acaagtcatc gtttgtgatc tgagacccgcggtgtggctg 1000 agagcgcaca gagcgcacgt gcacatacct ctgctagaaa ctcctgtcaa1050 ggcagcgaga gctgatgcac tcggacagag ctagacactc attcagaagc 1100ttttcgtttt ggccaaagtt gaccactact cttcttactc taacaagcca 1150 catgaatagaagaattttcc tcaagatgga cccggtaaat ataaccacaa 1200 ggaagcgaaa ctgggtgcgttcactgagtt gggttcctaa tctgtttctg 1250 gcctgattcc cgcatgagta ttagggtgatcttaaagagt ttgctcacgt 1300 aaacgcccgt gctgggccct gtgaagccag catgttcaccactggtcgtt 1350 cagcagccac gacagcacca tgtgagatgg cgaggtggct ggacagcacc1400 agcagcgcat cccggcggga acccagaaaa ggcttcttac acagcagcct 1450tacttcatcg gcccacagac accaccgcag tttcttctta aaggctctgc 1500 tgatcggtgttgcagtgtcc attgtggaga agctttttgg atcagcattt 1550 tgtaaaaaca accaaaatcaggaaggtaaa ttggttgctg gaagagggat 1600 cttgcctgag gaaccctgct tgtccaacagggtgtcagga tttaaggaaa 1650 accttcgtct taggctaagt ctgaaatggt actgaaatatgcttttctat 1700 gggtcttgtt tattttataa aattttacat ctaaattttt gctaaggatg1750 tattttgatt attgaaaaga aaatttctat ttaaactgta aatatattgt 1800catacaatgt taaataacct atttttttaa aaaagttcaa cttaaggtag 1850 aagttccaagctactagtgt taaattggaa aatatcaata attaagagta 1900 ttttacccaa ggaatcctctcatggaagtt tactgtgatg ttccttttct 1950 cacacaagtt ttagcctttt tcacaagggaactcatactg tctacacatc 2000 agaccatagt tgcttaggaa acctttaaaa attccagttaagcaatgttg 2050 aaatcagttt gcatctcttc aaaagaaacc tctcaggtta gctttgaact2100 gcctcttcct gagatgacta ggacagtctg tacccagagg ccacccagaa 2150gccctcagat gtacatacac agatgccagt cagctcctgg ggttgcgcca 2200 ggcgcccccgctctagctca ctgttgcctc gctgtctgcc aggaggccct 2250 gccatccttg ggccctggcagtggctgtgt cccagtgagc tttactcacg 2300 tggcccttgc ttcatccagc acagctctcaggtgggcact gcagggacac 2350 tggtgtcttc catgtagcgt cccagctttg ggctcctgtaacagacctct 2400 ttttggttat ggatggctca caaaataggg cccccaatgc tatttttttt2450 ttttaagttt gtttaattat ttgttaagat tgtctaaggc caaaggcaat 2500tgcgaaatca agtctgtcaa gtacaataac atttttaaaa gaaaatggat 2550 cccactgttcctctttgcca cagagaaagc acccagacgc cacaggctct 2600 gtcgcatttc aaaacaaaccatgatggagt ggcggccagt ccagcctttt 2650 aaagaacgtc aggtggagca gccaggtgaaaggcctggcg gggaggaaag 2700 tgaaacgcct gaatcaaaag cagttttcta attttgactttaaatttttc 2750 atccgccgga gacactgctc ccatttgtgg ggggacatta gcaacatcac2800 tcagaagcct gtgttcttca agagcaggtg ttctcagcct cacatgccct 2850gccgtgctgg actcaggact gaagtgctgt aaagcaagga gctgctgaga 2900 aggagcactccactgtgtgc ctggagaatg gctctcacta ctcaccttgt 2950 ctttcagctt ccagtgtcttgggtttttta tactttgaca gctttttttt 3000 aattgcatac atgagactgt gttgactttttttagttatg tgaaacactt 3050 tgccgcaggc cgcctggcag aggcaggaaa tgctccagcagtggctcagt 3100 gctccctggt gtctgctgca tggcatcctg gatgcttagc atgcaagttc3150 cctccatcat tgccaccttg gtagagaggg atggctcccc accctcagcg 3200ttggggattc acgctccagc ctccttcttg gttgtcatag tgatagggta 3250 gccttattgccccctcttct tataccctaa aaccttctac actagtgcca 3300 tgggaaccag gtctgaaaaagtagagagaa gtgaaagtag agtctgggaa 3350 gtagctgcct ataactgaga ctagacggaaaaggaatact cgtgtatttt 3400 aagatatgaa tgtgactcaa gactcgaggc cgatacgaggctgtgattct 3450 gcctttggat ggatgttgct gtacacagat gctacagact tgtactaaca3500 caccgtaatt tggcatttgt ttaacctcat ttataaaagc ttcaaaaaaa 3550 ccca3554 20 310 PRT Homo sapiens 20 Met Ala Leu Arg Arg Pro Pro Arg Leu ArgLeu Cys Ala Arg Leu 1 5 10 15 Pro Asp Phe Phe Leu Leu Leu Leu Phe ArgGly Cys Leu Ile Gly 20 25 30 Ala Val Asn Leu Lys Ser Ser Asn Arg Thr ProVal Val Gln Glu 35 40 45 Phe Glu Ser Val Glu Leu Ser Cys Ile Ile Thr AspSer Gln Thr 50 55 60 Ser Asp Pro Arg Ile Glu Trp Lys Lys Ile Gln Asp GluGln Thr 65 70 75 Thr Tyr Val Phe Phe Asp Asn Lys Ile Gln Gly Asp Leu AlaGly 80 85 90 Arg Ala Glu Ile Leu Gly Lys Thr Ser Leu Lys Ile Trp Asn Val95 100 105 Thr Arg Arg Asp Ser Ala Leu Tyr Arg Cys Glu Val Val Ala Arg110 115 120 Asn Asp Arg Lys Glu Ile Asp Glu Ile Val Ile Glu Leu Thr Val125 130 135 Gln Val Lys Pro Val Thr Pro Val Cys Arg Val Pro Lys Ala Val140 145 150 Pro Val Gly Lys Met Ala Thr Leu His Cys Gln Glu Ser Glu Gly155 160 165 His Pro Arg Pro His Tyr Ser Trp Tyr Arg Asn Asp Val Pro Leu170 175 180 Pro Thr Asp Ser Arg Ala Asn Pro Arg Phe Arg Asn Ser Ser Phe185 190 195 His Leu Asn Ser Glu Thr Gly Thr Leu Val Phe Thr Ala Val His200 205 210 Lys Asp Asp Ser Gly Gln Tyr Tyr Cys Ile Ala Ser Asn Asp Ala215 220 225 Gly Ser Ala Arg Cys Glu Glu Gln Glu Met Glu Val Tyr Asp Leu230 235 240 Asn Ile Gly Gly Ile Ile Gly Gly Val Leu Val Val Leu Ala Val245 250 255 Leu Ala Leu Ile Thr Leu Gly Ile Cys Cys Ala Tyr Arg Arg Gly260 265 270 Tyr Phe Ile Asn Asn Lys Gln Asp Gly Glu Ser Tyr Lys Asn Pro275 280 285 Gly Lys Pro Asp Gly Val Asn Tyr Ile Arg Thr Asp Glu Glu Gly290 295 300 Asp Phe Arg His Lys Ser Ser Phe Val Ile 305 310 21 3437 DNAHomo sapiens 21 caggaccagg tcttcctacg ctggagcagc ggggagacag ccaccatgca50 catcctcgtg gtccatgcca tggtgatcct gctgacgctg ggcccgcctc 100 gagccgacgacagcgagttc caggcgctgc tggacatctg gtttccggag 150 gagaagccac tgcccaccgccttcctggtg gacacatcgg aggaggcgct 200 gctgcttcct gactggctga agctgcgcatgatccgttct gaggtgctcc 250 gcctggtgga cgccgccctg caggacctgg agccgcagcagctgctgctg 300 ttcgtgcagt cgtttggcat ccccgtgtcc agcatgagca aactcctcca350 gttcctggac caggcagtgg cccacgaccc ccagactctg gagcagaaca 400tcatggacaa gaattacatg gcccacctgg tggaggtcca gcatgagcgc 450 ggcgcctccggaggccagac tttccactcc ttgctcacag cctccctgcc 500 gccccgccga gacagcacagaggcacccaa accaaagagc agcccagagc 550 agcccatagg ccagggccgg attcgggtggggacccagct ccgggtgctg 600 ggccctgagg acgacctggc tggcatgttc ctccagattttcccgctcag 650 cccggaccct cggtggcaga gctccagtcc ccgccccgtg gccctcgccc700 tgcagcaggc cctgggccag gagctggccc gcgtcgtcca gggcagcccc 750gaggtgccgg gcatcacggt gcgtgtcctg caggccctcg ccaccctgct 800 cagctccccacacggcggtg ccctggtgat gtccatgcac cgtagccact 850 tcctggcctg cccgctgctgcgccagctct gccagtacca gcgctgtgtg 900 ccacaggaca ccggcttctc ctcgctcttcctgaaggtgc tcctgcagat 950 gctgcagtgg ctggacagcc ctggcgtgga gggcgggcccctgcgggcac 1000 agctcaggat gcttgccagc caggcctcag ccgggcgcag gctcagtgat1050 gtgcgagggg ggctcctgcg cctggccgag gccctggcct tccgtcagga 1100cctggaggtg gtcagctcca ccgtccgtgc cgtcatcgcc accctgaggt 1150 ctggggagcagtgcagcgtg gagccggacc tgatcagcaa agtcctccag 1200 gggctgatcg aggtgaggtccccccacctg gaggagctgc tgactgcatt 1250 cttctctgcc actgcggatg ctgcctccccgtttccagcc tgtaagcccg 1300 ttgtggtggt gagctccctg ctgctgcagg aggaggagcccctggctggg 1350 gggaagccgg gtgcggacgg tggcagcctg gaggccgtgc ggctggggcc1400 ctcgtcaggc ctcctagtgg actggctgga aatgctggac cccgaggtgg 1450tcagcagctg ccccgacctg cagctcaggc tgctcttctc ccggaggaag 1500 ggcaaaggtcaggcccaggt gccctcgttc cgtccctacc tcctgaccct 1550 cttcacgcat cagtccagctggcccacact gcaccagtgc atccgagtcc 1600 tgctgggcaa gagccgggaa cagaggttcgacccctctgc ctctctggac 1650 ttcctctggg cctgcatcca tgttcctcgc atctggcaggggcgggacca 1700 gcgcaccccg cagaagcggc gggaggagct ggtgctgcgg gtccagggcc1750 cggagctcat cagcctggtg gagctgatcc tggccgaggc ggagacgcgg 1800agccaggacg gggacacagc cgcctgcagc ctcatccagg cccggctgcc 1850 cctgctgctcagctgctgct gtggggacga tgagagtgtc aggaaggtga 1900 cggagcacct gtcaggctgcatccagcagt ggggagacag cgtgctggga 1950 aggcgctgcc gagaccttct cctgcagctctacctacagc ggccggagct 2000 gcgggtgccc gtgcctgagg tcctactgca cagcgaaggggctgccagca 2050 gcagcgtctg caagctggac ggactcatcc accgcttcat cacgctcctt2100 gcggacacca gcgactcccg ggcgttggag aaccgagggg cggatgccag 2150catggcctgc cggaagctgg cggtggcgca cccgctgctg ctgctcaggc 2200 acctgcccatgatcgcggcg ctcctgcacg gccgcaccca cctcaacttc 2250 caggagttcc ggcagcagaaccacctgagc tgcttcctgc acgtgctggg 2300 cctgctggag ctgctgcagc cgcacgtgttccgcagcgag caccaggggg 2350 cgctgtggga ctgccttctg tccttcatcc gcctgctgctgaattacagg 2400 aagtcctccc gccatctggc tgccttcatc aacaagtttg tgcagttcat2450 ccataagtac attacctaca atgccccagc agccatctcc ttcctgcaga 2500agcacgccga cccgctccac gacctgtcct tcgacaacag tgacctggtg 2550 atgctgaaatccctccttgc agggctcagc ctgcccagca gggacgacag 2600 gaccgaccga ggcctggacgaagagggcga ggaggagagc tcagccggct 2650 ccttgcccct ggtcagcgtc tccctgttcacccctctgac cgcggccgag 2700 atggccccct acatgaaacg gctttcccgg ggccaaacggtggaggatct 2750 gctggaggtt ctgagtgaca tagacgagat gtcccggcgg agacccgaga2800 tcctgagctt cttctcgacc aacctgcagc ggctgatgag ctcggccgag 2850gagtgttgcc gcaacctcgc cttcagcctg gccctgcgct ccatgcagaa 2900 cagccccagcattgcagccg ctttcctgcc cacgttcatg tactgcctgg 2950 gcagccagga ctttgaggtggtgcagacgg ccctccggaa cctgcctgag 3000 tacgctctcc tgtgccaaga gcacgcggctgtgctgctcc accgggcctt 3050 cctggtgggc atgtacggcc agatggaccc cagcgcgcagatctccgagg 3100 ccctgaggat cctgcatatg gaggccgtga tgtgagcctg tggcagccga3150 cccccctcca agccccggcc cgtcccgtcc ccggggatcc tcgaggcaaa 3200gcccaggaag cgtgggcgtt gctggtctgt ccgaggaggt gagggcgccg 3250 agccctgaggccaggcaggc ccaggagcaa tactccgagc cctggggtgg 3300 ctccgggccg gccgctggcatcaggggccg tccagcaagc cctcattcac 3350 cttctgggcc acagccctgc cgcggagcggcggatccccc cgggcatggc 3400 ctgggctggt tttgaatgaa acgacctgaa ctgtcaa 343722 1029 PRT Homo sapiens 22 Met His Ile Leu Val Val His Ala Met Val IleLeu Leu Thr Leu 1 5 10 15 Gly Pro Pro Arg Ala Asp Asp Ser Glu Phe GlnAla Leu Leu Asp 20 25 30 Ile Trp Phe Pro Glu Glu Lys Pro Leu Pro Thr AlaPhe Leu Val 35 40 45 Asp Thr Ser Glu Glu Ala Leu Leu Leu Pro Asp Trp LeuLys Leu 50 55 60 Arg Met Ile Arg Ser Glu Val Leu Arg Leu Val Asp Ala AlaLeu 65 70 75 Gln Asp Leu Glu Pro Gln Gln Leu Leu Leu Phe Val Gln Ser Phe80 85 90 Gly Ile Pro Val Ser Ser Met Ser Lys Leu Leu Gln Phe Leu Asp 95100 105 Gln Ala Val Ala His Asp Pro Gln Thr Leu Glu Gln Asn Ile Met 110115 120 Asp Lys Asn Tyr Met Ala His Leu Val Glu Val Gln His Glu Arg 125130 135 Gly Ala Ser Gly Gly Gln Thr Phe His Ser Leu Leu Thr Ala Ser 140145 150 Leu Pro Pro Arg Arg Asp Ser Thr Glu Ala Pro Lys Pro Lys Ser 155160 165 Ser Pro Glu Gln Pro Ile Gly Gln Gly Arg Ile Arg Val Gly Thr 170175 180 Gln Leu Arg Val Leu Gly Pro Glu Asp Asp Leu Ala Gly Met Phe 185190 195 Leu Gln Ile Phe Pro Leu Ser Pro Asp Pro Arg Trp Gln Ser Ser 200205 210 Ser Pro Arg Pro Val Ala Leu Ala Leu Gln Gln Ala Leu Gly Gln 215220 225 Glu Leu Ala Arg Val Val Gln Gly Ser Pro Glu Val Pro Gly Ile 230235 240 Thr Val Arg Val Leu Gln Ala Leu Ala Thr Leu Leu Ser Ser Pro 245250 255 His Gly Gly Ala Leu Val Met Ser Met His Arg Ser His Phe Leu 260265 270 Ala Cys Pro Leu Leu Arg Gln Leu Cys Gln Tyr Gln Arg Cys Val 275280 285 Pro Gln Asp Thr Gly Phe Ser Ser Leu Phe Leu Lys Val Leu Leu 290295 300 Gln Met Leu Gln Trp Leu Asp Ser Pro Gly Val Glu Gly Gly Pro 305310 315 Leu Arg Ala Gln Leu Arg Met Leu Ala Ser Gln Ala Ser Ala Gly 320325 330 Arg Arg Leu Ser Asp Val Arg Gly Gly Leu Leu Arg Leu Ala Glu 335340 345 Ala Leu Ala Phe Arg Gln Asp Leu Glu Val Val Ser Ser Thr Val 350355 360 Arg Ala Val Ile Ala Thr Leu Arg Ser Gly Glu Gln Cys Ser Val 365370 375 Glu Pro Asp Leu Ile Ser Lys Val Leu Gln Gly Leu Ile Glu Val 380385 390 Arg Ser Pro His Leu Glu Glu Leu Leu Thr Ala Phe Phe Ser Ala 395400 405 Thr Ala Asp Ala Ala Ser Pro Phe Pro Ala Cys Lys Pro Val Val 410415 420 Val Val Ser Ser Leu Leu Leu Gln Glu Glu Glu Pro Leu Ala Gly 425430 435 Gly Lys Pro Gly Ala Asp Gly Gly Ser Leu Glu Ala Val Arg Leu 440445 450 Gly Pro Ser Ser Gly Leu Leu Val Asp Trp Leu Glu Met Leu Asp 455460 465 Pro Glu Val Val Ser Ser Cys Pro Asp Leu Gln Leu Arg Leu Leu 470475 480 Phe Ser Arg Arg Lys Gly Lys Gly Gln Ala Gln Val Pro Ser Phe 485490 495 Arg Pro Tyr Leu Leu Thr Leu Phe Thr His Gln Ser Ser Trp Pro 500505 510 Thr Leu His Gln Cys Ile Arg Val Leu Leu Gly Lys Ser Arg Glu 515520 525 Gln Arg Phe Asp Pro Ser Ala Ser Leu Asp Phe Leu Trp Ala Cys 530535 540 Ile His Val Pro Arg Ile Trp Gln Gly Arg Asp Gln Arg Thr Pro 545550 555 Gln Lys Arg Arg Glu Glu Leu Val Leu Arg Val Gln Gly Pro Glu 560565 570 Leu Ile Ser Leu Val Glu Leu Ile Leu Ala Glu Ala Glu Thr Arg 575580 585 Ser Gln Asp Gly Asp Thr Ala Ala Cys Ser Leu Ile Gln Ala Arg 590595 600 Leu Pro Leu Leu Leu Ser Cys Cys Cys Gly Asp Asp Glu Ser Val 605610 615 Arg Lys Val Thr Glu His Leu Ser Gly Cys Ile Gln Gln Trp Gly 620625 630 Asp Ser Val Leu Gly Arg Arg Cys Arg Asp Leu Leu Leu Gln Leu 635640 645 Tyr Leu Gln Arg Pro Glu Leu Arg Val Pro Val Pro Glu Val Leu 650655 660 Leu His Ser Glu Gly Ala Ala Ser Ser Ser Val Cys Lys Leu Asp 665670 675 Gly Leu Ile His Arg Phe Ile Thr Leu Leu Ala Asp Thr Ser Asp 680685 690 Ser Arg Ala Leu Glu Asn Arg Gly Ala Asp Ala Ser Met Ala Cys 695700 705 Arg Lys Leu Ala Val Ala His Pro Leu Leu Leu Leu Arg His Leu 710715 720 Pro Met Ile Ala Ala Leu Leu His Gly Arg Thr His Leu Asn Phe 725730 735 Gln Glu Phe Arg Gln Gln Asn His Leu Ser Cys Phe Leu His Val 740745 750 Leu Gly Leu Leu Glu Leu Leu Gln Pro His Val Phe Arg Ser Glu 755760 765 His Gln Gly Ala Leu Trp Asp Cys Leu Leu Ser Phe Ile Arg Leu 770775 780 Leu Leu Asn Tyr Arg Lys Ser Ser Arg His Leu Ala Ala Phe Ile 785790 795 Asn Lys Phe Val Gln Phe Ile His Lys Tyr Ile Thr Tyr Asn Ala 800805 810 Pro Ala Ala Ile Ser Phe Leu Gln Lys His Ala Asp Pro Leu His 815820 825 Asp Leu Ser Phe Asp Asn Ser Asp Leu Val Met Leu Lys Ser Leu 830835 840 Leu Ala Gly Leu Ser Leu Pro Ser Arg Asp Asp Arg Thr Asp Arg 845850 855 Gly Leu Asp Glu Glu Gly Glu Glu Glu Ser Ser Ala Gly Ser Leu 860865 870 Pro Leu Val Ser Val Ser Leu Phe Thr Pro Leu Thr Ala Ala Glu 875880 885 Met Ala Pro Tyr Met Lys Arg Leu Ser Arg Gly Gln Thr Val Glu 890895 900 Asp Leu Leu Glu Val Leu Ser Asp Ile Asp Glu Met Ser Arg Arg 905910 915 Arg Pro Glu Ile Leu Ser Phe Phe Ser Thr Asn Leu Gln Arg Leu 920925 930 Met Ser Ser Ala Glu Glu Cys Cys Arg Asn Leu Ala Phe Ser Leu 935940 945 Ala Leu Arg Ser Met Gln Asn Ser Pro Ser Ile Ala Ala Ala Phe 950955 960 Leu Pro Thr Phe Met Tyr Cys Leu Gly Ser Gln Asp Phe Glu Val 965970 975 Val Gln Thr Ala Leu Arg Asn Leu Pro Glu Tyr Ala Leu Leu Cys 980985 990 Gln Glu His Ala Ala Val Leu Leu His Arg Ala Phe Leu Val Gly 9951000 1005 Met Tyr Gly Gln Met Asp Pro Ser Ala Gln Ile Ser Glu Ala Leu1010 1015 1020 Arg Ile Leu His Met Glu Ala Val Met 1025 23 2186 DNA Homosapiens 23 ccgggccatg cagcctcggc cccgcgggcg cccgccgcgc acccgaggag 50atgaggctcc gcaatggcac cttcctgacg ctgctgctct tctgcctgtg 100 cgccttcctctcgctgtcct ggtacgcggc actcagcggc cagaaaggcg 150 acgttgtgga cgtttaccagcgggagttcc tggcgctgcg cgatcggttg 200 cacgcagctg agcaggagag cctcaagcgctccaaggagc tcaacctggt 250 gctggacgag atcaagaggg ccgtgtcaga aaggcaggcgctgcgagacg 300 gagacggcaa tcgcacctgg ggccgcctaa cagaggaccc ccgattgaag350 ccgtggaacg gctcacaccg gcacgtgctg cacctgccca ccgtcttcca 400tcacctgcca cacctgctgg ccaaggagag cagtctgcag cccgcggtgc 450 gcgtgggccagggccgcacc ggagtgtcgg tggtgatggg catcccgagc 500 gtgcggcgcg aggtgcactcgtacctgact gacactctgc actcgctcat 550 ctccgagctg agcccgcagg agaaggaggactcggtcatc gtggtgctga 600 tcgccgagac tgactcacag tacacttcgg cagtgacagagaacatcaag 650 gccttgttcc ccacggagat ccattctggg ctcctggagg tcatctcacc700 ctccccccac ttctaccctg acttctcccg cctccgagag tcctttgggg 750accccaagga gagagtcagg tggaggacca aacagaacct cgattactgc 800 ttcctcatgatgtacgcgca gtccaaaggc atctactacg tgcagctgga 850 ggatgacatc gtggccaagcccaactacct gagcaccatg aagaactttg 900 cactgcagca gccttcagag gactggatgatcctggagtt ctcccagctg 950 ggcttcattg gtaagatgtt caagtcgctg gacctgagcctgattgtaga 1000 gttcattctc atgttctacc gggacaagcc catcgactgg ctcctggacc1050 atattctgtg ggtgaaagtc tgcaaccccg agaaggatgc gaagcactgt 1100gaccggcaga aagccaacct gcggatccgc ttcaaaccgt ccctcttcca 1150 gcacgtgggcactcactcct cgctggctgg caagatccag aaactgaagg 1200 acaaagactt tggaaagcaggcgctgcgga aggagcatgt gaacccgcca 1250 gcagaggtga gcacgagcct gaagacataccagcacttca ccctggagaa 1300 agcctacctg cgcgaggact tcttctgggc cttcacccctgccgcggggg 1350 acttcatccg cttccgcttc ttccaacctc taagactgga gcggttcttc1400 ttccgcagtg ggaacatcga gcacccggag gacaagctct tcaacacgtc 1450tgtggaggtg ctgcccttcg acaaccctca gtcagacaag gaggccctgc 1500 aggagggccgcaccgccacc ctccggtacc ctcggagccc cgacggctac 1550 ctccagatcg gctccttctacaagggagtg gcagagggag aggtggaccc 1600 agccttcggc cctctggaag cactgcgcctctcgatccag acggactccc 1650 ctgtgtgggt gattctgagc gagatcttcc tgaaaaaggccgactaagct 1700 gcgggcttct gagggtaccc tgtggccagc cctgaagccc acatttctgg1750 gggtgtcgtc actgccgtcc ccggagggcc agatacggcc ccgcccaaag 1800ggttctgcct ggcgtcgggc ttgggccggc ctggggtccg ccgctggccc 1850 ggaggccctaggagctggtg ctgcccccgc ccgccgggcc gcggaggagg 1900 caggcggccc ccacactgtgcctgaggccc ggaaccgttc gcacccggcc 1950 tgccccagtc aggccgtttt agaagagcttttacttgggc gcccgccgtc 2000 tctggcgcga acactggaat gcatatacta ctttatgtgctgtgtttttt 2050 attcttggat acatttgatt ttttcacgta agtccacata tacttctata2100 agagcgtgac ttgtaataaa gggttaatga agaaaaaaaa aaaaaaaaaa 2150aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2186 24 548 PRT Homo sapiens 24Met Arg Leu Arg Asn Gly Thr Phe Leu Thr Leu Leu Leu Phe Cys 1 5 10 15Leu Cys Ala Phe Leu Ser Leu Ser Trp Tyr Ala Ala Leu Ser Gly 20 25 30 GlnLys Gly Asp Val Val Asp Val Tyr Gln Arg Glu Phe Leu Ala 35 40 45 Leu ArgAsp Arg Leu His Ala Ala Glu Gln Glu Ser Leu Lys Arg 50 55 60 Ser Lys GluLeu Asn Leu Val Leu Asp Glu Ile Lys Arg Ala Val 65 70 75 Ser Glu Arg GlnAla Leu Arg Asp Gly Asp Gly Asn Arg Thr Trp 80 85 90 Gly Arg Leu Thr GluAsp Pro Arg Leu Lys Pro Trp Asn Gly Ser 95 100 105 His Arg His Val LeuHis Leu Pro Thr Val Phe His His Leu Pro 110 115 120 His Leu Leu Ala LysGlu Ser Ser Leu Gln Pro Ala Val Arg Val 125 130 135 Gly Gln Gly Arg ThrGly Val Ser Val Val Met Gly Ile Pro Ser 140 145 150 Val Arg Arg Glu ValHis Ser Tyr Leu Thr Asp Thr Leu His Ser 155 160 165 Leu Ile Ser Glu LeuSer Pro Gln Glu Lys Glu Asp Ser Val Ile 170 175 180 Val Val Leu Ile AlaGlu Thr Asp Ser Gln Tyr Thr Ser Ala Val 185 190 195 Thr Glu Asn Ile LysAla Leu Phe Pro Thr Glu Ile His Ser Gly 200 205 210 Leu Leu Glu Val IleSer Pro Ser Pro His Phe Tyr Pro Asp Phe 215 220 225 Ser Arg Leu Arg GluSer Phe Gly Asp Pro Lys Glu Arg Val Arg 230 235 240 Trp Arg Thr Lys GlnAsn Leu Asp Tyr Cys Phe Leu Met Met Tyr 245 250 255 Ala Gln Ser Lys GlyIle Tyr Tyr Val Gln Leu Glu Asp Asp Ile 260 265 270 Val Ala Lys Pro AsnTyr Leu Ser Thr Met Lys Asn Phe Ala Leu 275 280 285 Gln Gln Pro Ser GluAsp Trp Met Ile Leu Glu Phe Ser Gln Leu 290 295 300 Gly Phe Ile Gly LysMet Phe Lys Ser Leu Asp Leu Ser Leu Ile 305 310 315 Val Glu Phe Ile LeuMet Phe Tyr Arg Asp Lys Pro Ile Asp Trp 320 325 330 Leu Leu Asp His IleLeu Trp Val Lys Val Cys Asn Pro Glu Lys 335 340 345 Asp Ala Lys His CysAsp Arg Gln Lys Ala Asn Leu Arg Ile Arg 350 355 360 Phe Lys Pro Ser LeuPhe Gln His Val Gly Thr His Ser Ser Leu 365 370 375 Ala Gly Lys Ile GlnLys Leu Lys Asp Lys Asp Phe Gly Lys Gln 380 385 390 Ala Leu Arg Lys GluHis Val Asn Pro Pro Ala Glu Val Ser Thr 395 400 405 Ser Leu Lys Thr TyrGln His Phe Thr Leu Glu Lys Ala Tyr Leu 410 415 420 Arg Glu Asp Phe PheTrp Ala Phe Thr Pro Ala Ala Gly Asp Phe 425 430 435 Ile Arg Phe Arg PhePhe Gln Pro Leu Arg Leu Glu Arg Phe Phe 440 445 450 Phe Arg Ser Gly AsnIle Glu His Pro Glu Asp Lys Leu Phe Asn 455 460 465 Thr Ser Val Glu ValLeu Pro Phe Asp Asn Pro Gln Ser Asp Lys 470 475 480 Glu Ala Leu Gln GluGly Arg Thr Ala Thr Leu Arg Tyr Pro Arg 485 490 495 Ser Pro Asp Gly TyrLeu Gln Ile Gly Ser Phe Tyr Lys Gly Val 500 505 510 Ala Glu Gly Glu ValAsp Pro Ala Phe Gly Pro Leu Glu Ala Leu 515 520 525 Arg Leu Ser Ile GlnThr Asp Ser Pro Val Trp Val Ile Leu Ser 530 535 540 Glu Ile Phe Leu LysLys Ala Asp 545 25 43 DNA Artificial Sequence Synthetic OligonucleotideProbe 25 tgtaaaacga cggccagtta aatagacctg caattattaa tct 43 26 41 DNAArtificial Sequence Synthetic Oligonucleotide Probe 26 caggaaacagctatgaccac ctgcacacct gcaaatccat t 41 27 19 DNA Artificial SequenceSynthetic Oligonucleotide Probe 27 actcgggatt cctgctgtt 19 28 23 DNAArtificial Sequence Synthetic Oligonucleotide Probe 28 aggcctttacccaaggccac aac 23 29 19 DNA Artificial Sequence SyntheticOligonucleotide Probe 29 ggcctgtcct gtgttctca 19 30 22 DNA ArtificialSequence Synthetic Oligonucleotide Probe 30 tcccaccact tacttccatg aa 2231 25 DNA Artificial Sequence Synthetic Oligonucleotide Probe 31ctgtggtacc caattgccgc cttgt 25 32 23 DNA Artificial Sequence SyntheticOligonucleotide Probe 32 attgtcctga gattcgagca aga 23 33 18 DNAArtificial Sequence Synthetic Oligonucleotide Probe 33 gtccagcaagccctcatt 18 34 20 DNA Artificial Sequence Synthetic OligonucleotideProbe 34 cttctgggcc acagccctgc 20 35 21 DNA Artificial SequenceSynthetic Oligonucleotide Probe 35 cagttcaggt cgtttcattc a 21 36 19 DNAArtificial Sequence Synthetic Oligonucleotide Probe 36 ccagtcaggccgttttaga 19 37 21 DNA Artificial Sequence Synthetic OligonucleotideProbe 37 cgggcgccca agtaaaagct c 21 38 28 DNA Artificial SequenceSynthetic Oligonucleotide Probe 38 cataaagtag tatatgcatt ccagtgtt 28

What is claimed is:
 1. Isolated nucleic acid having at least 80% nucleicacid sequence identity to a nucleotide sequence that encodes an aminoacid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6(SEQ ID NO:9), FIG. 8 (SEQ ID NO:11), FIG. 10 (SEQ ID NO:16), FIG. 12(SEQ ID NO:18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ ID NO:22), and FIG.18 (SEQ ID NO:24).
 2. Isolated nucleic acid having at least 80% nucleicacid sequence identity to a nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1),FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:8), FIG. 7 (SEQ ID NO:10), FIG.9 (SEQ ID NO:15), FIG. 11 (SEQ ID NO:17), FIG. 13 (SEQ ID NO:19), FIG.15 (SEQ ID NO:21) and FIG. 17 (SEQ ID NO:23).
 3. Isolated nucleic acidhaving at least 80% nucleic acid sequence identity to a nucleotidesequence selected from the group consisting of the full-length codingsequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG.3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:8), FIG. 7 (SEQ ID NO:10), FIG. 9(SEQ ID NO:15), FIG. 11 (SEQ ID NO:17), FIG. 13 (SEQ ID NO:19), FIG. 15(SEQ ID NO:21) and FIG. 17 (SEQ ID NO:23).
 4. Isolated nucleic acidhaving at least 80% nucleic acid sequence identity to the full-lengthcoding a sequence of the DNA deposited under ATCC accession number203538, 203661, 203583, 203657, 203576, 203573, 203553, 203651 and203537.
 5. A vector comprising the nucleic acid of any one of claims 1to
 4. 6. The vector of claim 5 operably linked to control sequencesrecognized by a host cell transformed with the vector.
 7. A host cellcomprising the vector of claim
 5. 8. The host cell of claim 7, whereinsaid cell is a CHO cell.
 9. The host cell of claim 7, wherein said cellis an E. coli.
 10. The host cell of claim 7, wherein said cell is ayeast cell.
 11. A process for producing a PRO polypeptides comprisingculturing the host cell of claim 7 under conditions suitable forexpression of said PRO polypeptide and recovering said PRO polypeptidefrom the cell culture.
 12. An isolated polypeptide having at least 80%amino acid sequence identity to an amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ IDNO:11), FIG. 10 (SEQ ID NO:16), FIG. 12 (SEQ ID NO:18), FIG. 14 (SEQ IDNO:20), FIG. 16 (SEQ ID NO:22), and FIG. 18 (SEQ ID NO:24).
 13. Anisolated polypeptide scoring at least 80% positives when compared to anamino acid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6(SEQ ID NO:9), FIG. 8 (SEQ ID NO:11), FIG. 10 (SEQ ID NO:16), FIG. 12(SEQ ID NO:18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ ID NO:22), and FIG.18 (SEQ ID NO:24).
 14. An isolated polypeptide having at least 80% aminoacid sequence identity to an amino acid sequence encoded by thefull-length coding sequence of the DNA deposited under ATCC accessionnumber 203538, 203661, 203583, 203657, 203576, 203573, 203553, 203651and
 203537. 15. A chimeric molecule comprising a polypeptide accordingto any one of claims 12 to 14 fused to a heterologous amino acidsequence.
 16. The chimeric molecule of claim 15, wherein saidheterologous amino acid sequence is an epitope tag sequence.
 17. Thechimeric molecule of claim 15, wherein said heterologous amino acidsequence is a Fc region of an immunoglobulin.
 18. An antibody whichspecifically binds to a polypeptide according to any one of claims 12 to14.
 19. The antibody of claim 18, wherein said antibody is a monoclonalantibody, a humanized antibody or a single-chain antibody.
 20. Isolatednucleic acid having at least 80% nucleic acid sequence identity to: (a)a nucleotide sequence encoding the polypeptide shown in FIG. 2 (SEQ IDNO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ IDNO:11), FIG. 10 (SEQ ID NO:16), FIG. 12 (SEQ ID NO:18), FIG. 14 (SEQ IDNO:20), FIG. 16 (SEQ ID NO:22), or FIG. 18 (SEQ ID NO:24), lacking itsassociated signal peptide; (b) a nucleotide sequence encoding anextracellular domain of the polypeptide shown in FIG. 2 (SEQ ID NO:2),FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID NO:11), FIG.10 (SEQ ID NO:16), FIG. 12 (SEQ ID NO:18), FIG. 14 (SEQ ID NO:20), FIG.16 (SEQ ID NO:22), or FIG. 18 (SEQ ID NO:24), with its associated signalpeptide; or (c) a nucleotide sequence encoding an extracellular domainof the polypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7),FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID NO:11), FIG. 10 (SEQ ID NO:16),FIG. 12 (SEQ ID NO:18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ ID NO:22),or FIG. 18 (SEQ ID NO:24), lacking its associated signal peptide.
 21. Anisolated polypeptide having at least 80% amino acid sequence identityto: (a) the polypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ IDNO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID NO:11), FIG. 10 (SEQ IDNO:16), FIG. 12 (SEQ ID NO:18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ IDNO:22), or FIG. 18 (SEQ ID NO:24), lacking its associated signalpeptide; (b) an extracellular domain of the polypeptide shown in FIG. 2(SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQID NO:11), FIG. 10 (SEQ ID NO:16), FIG. 12 (SEQ ID NO:18), FIG. 14 (SEQID NO:20), FIG. 16 (SEQ ID NO:22), or FIG. 18 (SEQ ID NO:24), with itsassociated signal peptide; or (c) an extracellular domain of thepolypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6(SEQ ID NO:9), FIG. 8 (SEQ ID NO:11), FIG. 10 (SEQ ID NO:16), FIG. 12(SEQ ID NO:18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ ID NO:22), or FIG.18 (SEQ ID NO:24), lacking its associated signal peptide.